Chronic exposure to trichloroethylene increases DNA methylation of the Ifng promoter in CD4+ T cells

Abstract

CD4⁺ T cells in female MRL+/+ mice exposed to solvent and water pollutant trichloroethylene (TCE) skew toward effector/memory CD4⁺ T cells, and demonstrate seemingly non-monotonic alterations in IFN-γ production. In the current study we examined the mechanism for this immunotoxicity using effector/memory and naïve CD4⁺ T cells isolated every 6 weeks during a 40 week exposure to TCE (0.5mg/ml in drinking water). A time-dependent effect of TCE exposure on both Ifng gene expression and IFN-γ protein production was observed in effector/memory CD4⁺ T cells, with an increase after 22 weeks of exposure and a decrease after 40 weeks of exposure. No such effect of TCE was observed in naïve CD4⁺ T cells. A cumulative increase in DNA methylation in the CpG sites of the promoter of the Ifng gene was observed in effector/memory, but not naïve, CD4⁺ T cells over time. Also unique to the Ifng promoter was an increase in methylation variance in effector/memory compared to naïve CD4⁺ T cells. Taken together, the CpG sites of the Ifng promoter in effector/memory CD4⁺ T cells were especially sensitive to the effects of TCE exposure, which may help explain the regulatory effect of the chemical on this gene.

Keywords: CD4(+) T cells; Epigenetics; Immunotoxicity; Next-generation sequencing; Trichloroethylene.

Figure 1. Chronic TCE exposure induced non-monotonic effects on IFN-γ

Female MRL+/+ mice were exposed to TCE (0.05 mg/ml) for 4, 22 or 40 weeks. Effector/memory (CD62L^lo) and naïve (CD62l^hi) CD4⁺ T cells were isolated from the spleens and activated ex vivo. Both Ifng expression (A) and secreted IFN-γ (B) was measured. *Significantly different (p<0.05) compared to control values. At the 40 week time point, the number of CD62L^lo effector/memory CD4⁺ T cells per mouse spleen was significantly different between control mice (5.6 ± 2.3 × 10⁷) and TCE-treated mice (8.9 ± 2 × 10⁷).

Figure 2. Relationship of methylation of CpG sites in the promoter and exon/intron of Ifng

Scatter plots show the correlation between the mean DNA methylation of CpG sites in the promoter in a single sample and the mean DNA methylation of the CpG sites in the exon/intron of the same sample. Data was collected from naïve and effector/memory CD4⁺ T cells across all time points, and each point represents a single sample.

Figure 3. Mean methylation of Ifng-associated CpG sites in both effector/memory and naïve CD4⁺ T cells

DNA methylation at CpG sites in both the promoter and exon/intron region of Ifng in effector/memory and naïve CD4⁺ T cells from control or TCE-treated mice was determined. Shown is the mean methylation of the data collected at all 7 time points. The CpG sites are designated by chromosome position. *Significantly different (p<0.05) compared to control values.

Figure 4. Alterations in methylation variance in CpG sites of Ifng promoter in effector/memory CD4⁺ T cells

Methylation variance was measured across all time points for CpG sites in the promoter or exon/intron regions of Ifng in both naïve and effector/memory CD4⁺ T cells from control and TCE-treated mice. *Significantly different (p<0.05) compared to control values.