Prevention of Adult Colitis by Oral Ferric Iron in Juvenile Mice Is Associated with the Inhibition of the Tbet Promoter Hypomethylation and Gene Overexpression

Abstract

Iron is an essential nutrient needed for physiological functions, particularly during the developmental period of the early childhood of at-risk populations. The purpose of this study was to investigate, in an experimental colitis, the consequences of daily oral iron ingestion in the early period on the inflammatory response, the spleen T helper (Th) profiles and the associated molecular mechanisms. Juvenile mice orally received microencapsulated ferric iron or water for 6 weeks. On adult mice, we induced a sham or experimental trinitrobenzene sulfonic acid (TNBS) moderate colitis during the last week of the experiment before sacrificing the animals 7 days later. The severity of the gut inflammation was assessed by macroscopic damage scores (MDS) and the myeloperoxidase activity (MPO). Th profiles were evaluated by the examination of the splenic gene expression of key transcription factors of the Th differentiation (Tbet, Gata3, Foxp3 and RORγ) and the methylation of their respective promoter. While TNBS-induced colitis was associated with a change of the Th profile (notably an increase in the Tbet/Gata3 ratio in the spleen), the colitis-inhibition induced by ferric iron was associated with a limitation of the splenic Th profiles perturbation. The inhibition of the splenic Tbet gene overexpression was associated with an inhibition of promoter hypomethylation. In summary, mice treated by long-term oral ferric iron in the early period of life exhibited an inhibition of colitis associated with the inhibition of the splenic Tbet promoter hypomethylation and gene overexpression.

Keywords: Tbet; Th profile; ferric iron; intestinal inflammation; promoter methylation.

Figure 1

The experimental design of the 6-week-long study (W1 to W6). Animals received 200 µL/day of either ferric iron (75 or 150 mg/kg/day po) or water during the 6 weeks. Animals received trinitrobenzene sulfonic acid (TNBS) (100 mg/kg) or its vehicle during week 6 and were kept for seven days before sacrifice.

Figure 2

The representation of the promotor genomic regions of Tbet and Gata3 showing the localization of all CpG regions present into promotor regions of these transcription factors.

Figure 3

The impact of iron supplementation on the Gata3 and Tbet gene expression ratio in the spleen. Data are expressed as Mean ± SEM. *** Significantly different (p < 0.001) from vehicle-treated TNBS mice. +++ Significantly different (p < 0.001) from vehicle-treated control mice. White column: Control mice, Black column: TNBS-treated mice. TNBS instillation resulted in an increase of the Tbet/Gata3 ratio. By contrast, ferric iron ingestion dose-dependently limited the Tbet/Gata3 ratio increase in TNBS-treated mice.

Figure 4

The impact of iron supplementation on the Tbet (a), Gata3 (b), RORɣ (c) and Foxp3 (d) gene expressions in the spleen. Data are expressed as a Tukey boxplot with a median. Each small black dot (•) represents individual data that is not included between the whiskers. **, *** significantly different (p < 0.01 and p < 0.001, respectively) from the controls (CTL). +, ++, +++ significantly different (p < 0.05, p < 0.01 and p < 0.001, respectively) from the vehicle-treated TNBS mice (veh-TNBS). Groups: Controls (CTL), Vehicle-treated TNBS mice (veh-TNBS), Ferric iron (75 mg/kg/day po), TNBS-treated mice (Iron 75-TNBS), Ferric iron (150 mg/kg/day po) TNBS-treated mice (Iron 150-TNBS). Tbet, RORɣ and Foxp3 are significantly increased in TNBS treated mice (a,c,d). In contrast, splenic gene expression of Tbet (a), RORɣ (c) and Foxp3 (d) were significantly lower in mice supplemented by ferric iron. Splenic Gata3 expression was also enhanced in mice treated with ferric iron (150 mg/kg/day po).

Figure 5

The impact of ferric iron supplementation on the Tbet (a) and Gata3 (b) promoter CpG methylation status (%). Data are expressed as Mean ± SEM. * Significantly different (p < 0.05) from vehicle-treated mice. ++ Significantly different (p < 0.01) from vehicle-treated TNBS mice. White columns: vehicle-treated mice; squared columns: ferric iron (150 mg/kg/g po) treated mice. TNBS instillation resulted in a significant reduction (p < 0.001) of the Tbet promoter CpG methylation status (A) which was reversed by iron treatment (a). By contrast, we did not observe any modification of the methylation status of the promoter region of Gata3 (b) regardless of whichever treatment was considered.