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. 2017 Apr 21;7(1):1007.
doi: 10.1038/s41598-017-01249-7.

Hypermethylation of Interferon Regulatory Factor 8 (IRF8) Confers Risk to Vogt-Koyanagi-Harada Disease

Yiguo Qiu  1 , Hongsong Yu  1 , Yunyun Zhu  1 , Zi Ye  1 , Jing Deng  1 , Wencheng Su  1 , Qingfeng Cao  1 , Gangxiang Yuan  1 , Aize Kijlstra  2 , Peizeng Yang  3
Affiliations

Hypermethylation of Interferon Regulatory Factor 8 (IRF8) Confers Risk to Vogt-Koyanagi-Harada Disease

Yiguo Qiu et al. Sci Rep. .

Abstract

Aberrant methylation change of IRF8 confers risk to various tumors, and abnormal expression of IRF8 is involved in many autoimmune diseases, including ocular Behcet's disease. However, whether the methylation change of IRF8 is associated with Vogt-Koyanagi-Harada (VKH) disease remains unknown. In the present study, we found a decreased IRF8 mRNA expression in association with a higher methylation level in monocyte-derived dendritic cells (DCs) from active VKH patients compared with the normal and inactive subjects. DCs incubated with cyclosporin a (CsA) or dexamethasone (DEX) showed a lower methylation and higher mRNA expression of IRF8 in active VKH patients. A demethylation reagent, 5-Aza-2'-deoxycytidine (DAC) showed a notable demethylation effect as evidenced by increasing the mRNA expression and reducing the methylation level of IRF8. It also suppressed the Th1 and Th17 responses through down-regulating the expression of co-stimulatory molecules (CD86, CD80, CD40), and reducing the production of pro-inflammatory cytokines (IL-6, IL-1β, IL-23, IL-12) produced by DCs. These findings shows that hypermethylation of IRF8 in DCs confers risk to VKH disease. Demethylation of IRF8 may offer a novel therapeutic strategy protect against VKH disease.

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Conflict of interest statement

The authors declare that they have no competing interests.

Figures

Figure 1
Figure 1
A lower mRNA expression and a higher methylation level of IRF8 in the DCs of active VKH patients is detected than normal controls. (A) DCs from normal controls and active VKH patients were cultured and collected to detect the expression of IRF8 with real-time PCR (normal controls: n = 9; VKH patients: n = 7, **p < 0.01). Methylation levels of the CpG sites between −441 and −225 from TSS of the first exon were detected with matrix assisted laser desorption ionization time-of-flight (MALDI-TOF) mass spectrometer in the DCs of VKH patients and normal controls. The methylation changes shown in CpG_1 (B), CpG_7.8 (C) and CpG_16 (D) of VKH patients were increased compared with normal controls (VKH, n = 20; normal controls, n = 30, *p < 0.05, **p < 0.01). The data are shown as mean ± SEM. All the values were normally distributed. Unpaired t test was used to compare the methylation change of a certain CpG site between two groups.
Figure 2
Figure 2
The methylation and the mRNA expression changes of IRF8 affect the activity of VKH patients. (A) DCs from inactive and active VKH patients were cultured and collected to detect the expression of IRF8 with real-time PCR (n = 10, *p < 0.05). The methylation changes shown in CpG_1 (B), CpG_7.8 (C) and CpG_16 (D) of inactive VKH patients were notably lower than active patients (n = 10, **p < 0.01, ***p < 0.001). The data are shown as mean ± SEM. Unpaired t test was used to compare the mRNA level and methylation changes between two groups.
Figure 3
Figure 3
CsA and DEX treatment affects the methylation change and mRNA expression of IRF8 in active VKH patients. (A) The mRNA expression of IRF8 was detected by real-time PCR in DCs with or without the treatment with CsA or DEX. DCs were obtained from active VKH patients (n = 8, *p < 0.05). The methylation changes shown in CpG_1 (B), CpG_7.8 (C) and CpG_16 (D) of CsA treated and DEX treated DCs were significantly decreased compared to the untreated DCs from active VKH patients (n = 8, *p < 0.05, **p < 0.01, ***p < 0.001). The data are shown as mean ± SEM. One way-ANOVA, followed by Bonferroni correction was used to compare the mRNA level and methylation changes among multiple groups.
Figure 4
Figure 4
DAC treatment shows a demethylation effect and restores the mRNA expression of IRF8. (A) DAC treated and untreated DCs from active VKH patients were collected to detect the expression of IRF8 with real-time PCR (VKH, n = 12, VKH + DAC, n = 12, ***p < 0.001). The methylation changes in CpG_1 (B), CpG_7.8 (C) and CpG_16 (D) of DAC treated and untreated DCs from VKH patients were evaluated with MALDI-TOF mass spectrometer (VKH, n = 10; VKH + DAC, n = 10, *p < 0.05, **p < 0.01). The data are shown as mean ± SEM. Paired-t test was used to compare the methylation changes between two groups.
Figure 5
Figure 5
DAC reduces the expression of surface markers of DCs from VKH patients. Immature DCs from VKH patients were cultured with or without the presence of DAC for 6 days, then followed by stimulation with 100 ng/mL LPS for 24 hours. DCs were subsequently harvested for flow cytometry analysis with specific surface antibodies against CD80, HLA-DR, CD86, CD83 and CD40. (A) Histograms with overlays are from representative experiments of DAC treated and untreated DCs from VKH patients. (B) Mean fluorescence intensity (MFI) of surface markers on DAC treated and untreated DCs from VKH patients. Data are shown as mean ± SEM. Statistical analysis was performed using the paired-samples t-test (n = 8, *p < 0.05; **p < 0.01).
Figure 6
Figure 6
DAC affects the production of inflammatory cytokines by DCs from VKH patients. CD14+ monocytes from VKH patients and normal controls were cultured for 6 days and then stimulated with 100 ng/ml LPS for 24 hours. The protein concentrations of IL-6, IL-1β, IL-23 and IL-12p70 in the supernatants were determined by ELISA. (A) The expression of IL-6, IL-1β, IL-23 and IL-12p70 was significantly increased in VKH patients compared with normal controls (n = 12, *p < 0.05,**p < 0.01, ***p < 0.001). (B) The protein concentrations of IL-6, IL-1β, IL-23 and IL-12p70 were reduced if cells (from VKH patients) were pretreated with 10 µM of DAC (n = 12, *p < 0.05). The data are shown as mean ± SEM. The paired-samples t-test was performed for the statistical analysis.
Figure 7
Figure 7
DAC treatment inhibits the Th1 and Th17 responses. Allogeneic CD4+ T cells (obtained from healthy controls) were co-cultured with DAC-treated or –untreated DCs (derived from VKH patients) for 5 days. The frequencies of IFN-γ+ (A) and IL-17+ cells (B) in the CD4+ T cells co-cultured with DAC-treated or –untreated DCs were evaluated by flow cytometry (n = 7, *p < 0.05,**p < 0.01). (C) The representative results of the IFN-γ+ and IL-17+ cells in the CD4+ T cells co-cultured with DAC-treated or –untreated DCs. The protein levels of IFN-γ (D) and IL-17 (E) in the cell culture supernatants were measured by ELISA (n = 7, *p < 0.05). The data are shown as mean ± SEM. Ctr-DCs: control DCs (DAC-untreated DCs), DAC-DCs: DAC-treated DCs. Paired t test was used to compare the protein concentrations of IFN-γ and IL-17 and the frequencies of IFN-γ+ and IL-17+ cells between two groups.
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