Abstract
Purpose :
Age related macular degeneration (AMD) is a degenerative disease of the central retina and the leading cause of irreversible blindness worldwide. We previously reported that CCL26 (eotaxin-3), an inflammatory chemokine, is epigenetically regulated by histone deacetylase-1/2 (HDAC1/2) in human retinal pigment epithelium (hRPE). In this study, we evaluated the expression of CCL11/24/26 (eotaxins-1/2/3) in eyes with atrophic AMD compared to age-matched controls and examined epigenetic regulation of CCL26 in targeted knockout (KO) models of HDAC1/2.
Methods :
CCL11/24/26 levels in RPE/choroid tissues of advanced dry AMD patients (n=5) and age-matched controls (n=5) were determined using qPCR and immunohistochemistry (IHC). Viability of hRPE cells (n=3) was evaluated after treatment with different doses of recombinant CCL26 (10, 100, and 1000 ng/ml) for 48h followed by SYTOX staining. RNA samples and cell lysates of HDAC1/2 KO hRPE models generated using short-interfering RNA (siRNA) and CRISPR/Cas9 based approaches were analyzed using qPCR and multiplex bead arrays. HDAC1/2 interaction with the CCL26 promoter was observed using chromatin immunoprecipitation assay. Statistical testing was done (p-value <0.05) using the Mann-Whitney U test.
Results :
Posterior segment showed high expression of CCL26, followed by CCL24 and low levels of CCL11. CCL26 mRNA was significantly increased in RPE/choroid (~7-fold change) in atrophic AMD patients compared to controls. IHC in whole eye sections revealed moderate CCL26 expression in the ganglion cell layer (GCL), inner nuclear layer (INL), outer nuclear layer (ONL) in both control and atrophic AMD eyes. However, in atrophic AMD eyes, elevated CCL26 deposition localized to RPE/Bruch’s membrane. Human RPE cells treated with CCL26 demonstrated dose-dependent cytotoxicity. HDAC1/2 KO models showed increased CCL26 expression compared to controls. ChIP assay revealed enhanced binding of HDAC1/2 to CCL26 promoter which was blocked by a pan-HDAC inhibitor, valproic acid.
Conclusions :
CCL26 expression in RPE/choroid is significantly elevated in atrophic AMD eyes compared to controls and is epigenetically regulated by HDAC1/2. Our results suggest that CCL26 may play a crucial role in the RPE cell death process observed in atrophic AMD. Collectively, our data suggest that selective neutralization of CCL26 may be therapeutically effective in the treatment of atrophic AMD.
This abstract was presented at the 2022 ARVO Annual Meeting, held in Denver, CO, May 1-4, 2022, and virtually.