Abstract
Purpose :
The retinal pigment epithelia (RPE) secrete inflammatory cytokines such as Interferon (IFN) during Age-related macular degeneration (AMD), but the molecular mechanisms for this are not understood. We previously reported that double-stranded RNA (dsRNA) can induce IFN secretion and RPE degeneration through RIG-I signaling. The goal of this study is to explore the biological processes of dsRNA accumulation in AMD pathobiology.
Methods :
Postmortem human eyes were obtained from the Lions Eye Institute for transplant & Research (LEITR; Tampa Fl). Immunohistochemistry (IHC) was performed using a dsRNA specific antibody, J2 (Scicons), comparing AMD to non-AMD donor eyes. iPS-RPE cells (FujiFilm) were treated with inhibitors to pathways predicted to regulate dsRNA for 3d at non-cytotoxic doses, then assessed for dsRNA through immunostaining and IFNβ secretion using ELISA. Transepithelial resistance (TER) was measured using cellZscope. Probes against known dsRNAs, HERV and Alu (ThermoFisher), were used to assess transcription in treated cells via RT-qPCR.
Results :
IHC for dsRNA found that RPE stain positively in samples from AMD patients, increasing with severity. Treating iPS-RPE cells with inhibitors against pathways predicted to regulate dsRNA revealed that inhibition of histone deacetylase (HDAC) with Trichostatin A (TSA) induces dsRNA accumulation and IFNβ secretion (DMSO - 1.5pg/ml vs TSA - 11.6pg/ml, p<0.005). TSA also induces complete loss of iPS-RPE barrier function within 3d. We hypothesized that TSA-induced dsRNA accumulation in RPE to be caused by transcription of dsRNA entities such as endogenous retrovirus elements (ERV) and retrotransposons. Associated transcripts were assessed via qPCR after TSA treatment, and found increased compared to controls (TSA vs. Control HERV = 4.2±0.05 fold changes, p<0.005; ALU = 2.0±0.16 fold changes, p<0.005).
Conclusions :
dsRNA accumulates in RPE of AMD donor eyes. We found TSA treatment to increase transcription of dsRNAin iPS-RPE cells resulting in IFN secretion, and barrier function loss. We conclude that impairment of HDAC function can induce dsRNA accumulation in RPE in vitro and may be a useful tool for understanding the role of dsRNA in RPE health and disease.
This abstract was presented at the 2024 ARVO Annual Meeting, held in Seattle, WA, May 5-9, 2024.