DNMT1-mediated Foxp3 gene promoter hypermethylation involved in immune dysfunction caused by arsenic in human lymphocytes

Abstract

Growing evidence indicates that arsenic can cause long-lasting and irreversible damage to the function of the human immune system. It is known that forkhead box protein 3(Foxp3), which is specifically expressed in regulatory T cells (Tregs), plays a decisive role in immunoregulation and is regulated by DNA methylation. While evidence suggests that epigenetic regulated Foxp3 is involved in the immune disorders caused by arsenic exposure, the specific mechanism remains unclear. In this study, after primary human lymphocytes were treated with different doses of NaAsO₂, our results showed that arsenic induced the high expression of DNMT1 and Foxp3 gene promoter methylation level, thereby inhibiting the expression levels of Foxp3, followed by decreasing Tregs and reducing related anti-inflammatory cytokines, such as interleukin 10 (IL-10) and interleukin 10 (IL-35), and increasing the ratio of CD4⁺/CD8⁺ T cells in lymphocytes. Treatment with DNA methyltransferase inhibitor 5-Aza-CdR can notably inhibit the expression of DNMT1, effectively restoring the hypermethylation of the Foxp3 promoter region in primary human lymphocytes and upregulating the expression levels of Foxp3, balancing the ratio of CD4⁺/CD8⁺ T cells in lymphocytes. It also activates the secretion of anti-inflammatory cytokines and restores the immune regulatory functions of Tregs. In conclusion, our study provides limited evidence that DNMT1-mediated Foxp3 gene promoter hypermethylation is involved in immune dysfunction caused by arsenic in primary human lymphocytes. The study can provide a scientific basis for further understanding the arsenic-induced immune dysfunction in primary human lymphocytes.

Keywords: 5-Aza-CdR; DNA methylation; DNMT1; Foxp3; arsenic; lymphocytes.

Figure 1

Arsenic-induced immune dysfunction in human lymphocytes by altering the level of DNMT1-mediated Foxp3 promoter methylation. Lymphocytes were treated with different concentrations of NaAsO₂ for 24 h. (A) Cell viability changes of human lymphocytes exposed to NaAsO₂ and detected by MTT assay. The expression levels of Foxp3 mRNA (B) and protein (C) in lymphocyte exposure to NaAsO₂. Transcription (D) and translation (E) levels of DNMT1 in lymphocytes exposed to NaAsO₂. (F) Western blot and analysis of densitometric indicate the expression level of DNMT1 and Foxp3 proteins. (G) Promoter methylation levels of Foxp3 genes in lymphocytes exposed to NaAsO₂. (H) Enzyme-linked immunosorbent assay of IL-10 and IL-35 expression levels from lymphocyte supernatant exposed to different dosages of NaAsO₂. ^*Significant changes with respect to the control group, P < 0.05. The results were expressed as mean ± SD of three independent experiments.

Figure 2

Alterations of T lymphocyte subpopulation in primary human lymphocytes by arsenic exposure. CD4⁺ and CD8⁺ T cells in lymphocytes (A) are shown. Quantitative analysis of CD4⁺ (C) and CD8⁺ (D) T cells in lymphocytes and CD4⁺/CD8⁺ ratio in lymphocytes (E) were calculated. The results of flow cytometer detection of Tregs in CD4⁺ T cells (B) exhibit a dose-dependent decline tendency with the concentration of NaAsO₂. The percentage of Tregs in CD4⁺ T lymphocytes after exposed to different concentrations of NaAsO₂ is calculated in (F). ^*Significant changes with respect to the control group, P < 0.05. The results were expressed as mean ± SD of three independent experiments.

Figure 3

Treatment with 5-Aza-CdR can effectively restore the immune dysfunction induced by arsenic in human lymphocytes. Lymphocytes were treated with NaAsO₂ (20.00 μmol/l) for 24 h followed by 5-Aza-CdR (5.00 μmol/l) for 72 h. (A) The expression levels of Foxp3 and DNMT1 protein after treated with 5-Aza-CdR. (B) The levels of Foxp3 gene promoter methylation after treated with 5-Aza-CdR. (C) The expression levels of Treg-related cytokines (IL-10, IL-35) in lymphocyte culture supernatant were estimated after treated with 5-Aza-CdR. The ratio of Treg in human CD4⁺ T lymphocytes treated with NaAsO₂ or 5-Aza-CdR alone and co-exposed. (D) The expression level of proteins expressed by western blot. ^*Compared with control group, P < 0.05. ^#Compared with the NaAsO₂ treated alone, P < 0.05. Mean ± SD was used to express all the data of three independent experiments.

Figure 4

Treatment of 5-Aza-CdR can alleviate the disorder of T lymphocyte subpopulation after exposure of arsenic in primary human lymphocytes. CD4⁺ and CD8⁺ T cells in lymphocytes (A) are shown. Quantitative analysis of CD4⁺ (C) and CD8⁺ (D) T cells in lymphocytes, and CD4⁺/CD8⁺ ratio in lymphocytes (E) were calculated. Tregs were measured by flow cytometer (B) and the ratio of Tregs in CD4⁺ T lymphocytes after treated with 5-Aza-CdR was calculated in (F). ^*Compared with control group, P < 0.05. ^#Compared with the NaAsO₂ treated alone, P < 0.05. Mean ± SD was used to express all the data of three independent experiments.