Dynamic DNA Methylation Changes of Tbx21 and Rorc during Experimental Autoimmune Uveitis in Mice

Abstract

The key transcription factors of T helper cell subpopulations, including T-bet, GATA3, RORγt, and Foxp3 are involved in various autoimmune diseases. Whether methylation of these master transcription factors is associated with the development of experimental autoimmune uveitis (EAU) and the possible epigenetic regulatory mechanisms involved has however not yet been addressed. In our study, significant methylation changes in both Tbx21 and Rorc were observed in one CpG site in the retinas of EAU mice. Two CpG sites of Tbx21 and one CpG site of Rorc showed significant dynamic methylation changes in the RPE-choroid complex during EAU. The mRNA expressions of Tbx21 and Rorc in both the retinas and RPE-choroid complexes correlated with the methylation changes at the various time points during EAU development. The methylation changes were associated with the production of the Th1/Th17 cells' signature cytokines, IFN-γ and IL-17. Dynamic changes in mRNA expression of DNA methyltransferases (DNMT1) were also noted, which may be related to the observed methylation changes of these genes. The present study provides evidence that DNA methylation of Tbx21 and Rorc may be associated with the development of EAU. DNMT1 activation may have an important effect on regulating DNA methylation dynamics.

Figure 1

The methylation level of Tbx21 and Rorc in the retinas and RPE-choroid complexes of EAU mice at different time points. The retinal methylation changes of CpG_1 for Tbx21 (a) and Rorc (b) at different time points during EAU were detected with a MALDI-TOF mass spectrometer (n = 6, ^∗ p < 0.05, ^∗∗ p < 0.01, and ^∗∗∗ p < 0.001). In the RPE-choroid complexes, the methylation levels of Tbx21 in CpG_1 (c) and CpG_5 (d) and Rorc in CpG_5 (e) also showed a dynamic alteration with statistical difference (^∗ p < 0.05, ^∗∗ p < 0.01, ^∗∗∗ p < 0.001, and n = 6). The data are shown as mean ± SEM. One-way ANOVA followed by Bonferroni correction was used to compare the methylation levels in multiple groups.

Figure 2

The mRNA expression of Tbx21 and Rorc in the retinas and RPE-choroid complexes of EAU mice at different time points. Both the retinas and RPE-choroid complexes were dissected at different time points following IRBP peptide immunization. Then mRNA expressions of Tbx21 and Rorc in the retinas (a, b) and RPE-choroid complexes (c, d) were detected with real-time PCR (^∗ p < 0.05, ^∗∗ p < 0.01, ^∗∗∗ p < 0.001, and n = 6). The data are shown as mean ± SEM. One-way ANOVA followed by Bonferroni correction was used to correct for multiple comparisons.

Figure 3

Correlation between the methylation levels of significantly changed CpG sites and the mRNA expression of Tbx21 and Rorc in the retinas of EAU mice at different time points. The methylation levels of CpG-1 in Tbx21 (a) and CpG-1 in Rorc (b) were negatively correlated with their mRNA expression in the retinas of the mice. Pearson correlation test was used to perform the correlation analysis between the significantly changed methylation CpG sites and the mRNA expression levels of Tbx21 and Rorc.

Figure 4

Correlation between the methylation levels of significantly changed CpG sites and the mRNA expression of Tbx21 and Rorc in the RPE-choroid complexes of EAU mice at different time points. The methylation level of CpG-1 and CpG-5 in Tbx21 (a, b) and CpG-5 in Rorc (c) were negatively correlated with their mRNA expression in the RPE-choroid complexes of the mice. Pearson correlation test was used to perform the correlation analysis between the significantly changed methylation CpG sites and the mRNA expression levels of Tbx21and Rorc.

Figure 5

The mRNA expression of IFN-γ and IL-17 in the retinas and RPE-choroid complexes of EAU mice at different time points. The mRNA expressions of IFN-γ and IL-17 in the retinas (a, b) and RPE-choroid complexes (c, d) were detected with real-time PCR at different time points. (^∗ p < 0.05, ^∗∗ p < 0.01, ^∗∗∗ p < 0.001, and n = 6). The data are shown as mean ± SEM. One-way ANOVA followed by Bonferroni correction was applied to compare the mRNA expressions in multiple groups.

Figure 6

The mRNA expression of DNMT1, DNMT3a, and DNMT3b in the retina and RPE-choroid complex of EAU mice at different time points. Total RNA was extracted from the retinas and RPE-choroid complexes of EAU mice at different time points. The mRNA expression of DNMT1, DNMT3a, and DNMT3b in the retina (a) and RPE-choroid complex (b) was detected with real-time PCR (^∗ p < 0.05, ^∗∗ p < 0.01, ^∗∗∗ p < 0.001, and n = 6). The data are shown as mean ± SEM. One-way ANOVA followed by Bonferroni correction was used to analyze the mRNA expressions.