Hypermethylation of Interferon Regulatory Factor 8 (IRF8) confers risk to Vogt-Koyanagi-Harada Disease

Abstract

Purpose : To investigate the methylation change of IRF8 in monocyte-derived dendritic cells (DCs) obtained from Vogt-Koyanagi-Harada (VKH) patients, and in the DCs incubated with conventional drugs, thus to study the effect of IRF8 demethylation on the in vitro function of DCs, and Th1/Th17 cell responses in patient group.

Methods : Monocyte-derived DCs from VKH patients were cultured with or without the presence of 5-Aza-2′-deoxycytidine (DAC), cyclosporin a (CsA) or dexamethasone (DEX). The mRNA expression of IRF8 was determined by real-time PCR. The methylation level of IRF8 promoter was detected by mass spectrometry. The demethylation effect of DAC on DCs and on Th1/Th17 responses was evaluated by ELISA and flow cytometry. Two-tailed Student's t test, Wilcoxon’s matched-pairs test or paired-samples t-test were used for the statistical analysis.

Results : A decreased IRF8 mRNA expression in association with a higher methylation level was observed in active VKH patients compared to controls (*p<0.05, **p<0.01). Inactive VKH patients that responded to treatments showed a down-regulated methylation level and increased mRNA expression of IRF8 compared to active VKH patients (*p<0.05, **p<0.01, ***p<0.001). The DCs from active untreated VKH patients which were incubated with CsA or DEX also showed a lower methylation and higher mRNA expression of IRF8 than untreated DCs (*p<0.05, **p<0.01, ***p<0.001). DAC showed a notable demethylation effect as evidenced by increasing the mRNA expression and reducing the methylation level of IRF8 (*p<0.05, **p<0.01, ***p<0.001). It also suppressed the Th1 and Th17 responses through down-regulating the expression of co-stimulatory molecules (CD86, CD80, and CD40), and reducing the expression of pro-inflammatory cytokines ( IL-6, IL-1β, IL-23 and IL-12) secreted by DCs (*p<0.05, **p<0.01, ***p<0.001). The frequencies of Th1, Th17 cells and the production of IFN-γ, IL-17 were also decreased with the treatment of DAC (*p<0.05, **p<0.01).

Conclusions : These findings show that hypermethylation of IRF8 in DCs confers risk to VKH disease. Demethylation of IRF8 may offer a novel therapeutic opportunity in the treatment of VKH disease.

This is an abstract that was submitted for the 2017 ARVO Annual Meeting, held in Baltimore, MD, May 7-11, 2017.