Epigenetic modification of FOXP3 in patients with chronic HIV infection

Abstract

Objectives: HIV-1 modulates host cell epigenetic machinery to control its own replication and induce immune suppression. HIV-1 infection leads to activation of T regulatory cell (T(reg)), but the mechanism underlying this immune modulation is unclear. T(reg) plays a prominent role in gut-mucosal immune tolerance by restraining excessive effector T-cell responses, a mechanism that is known to be disturbed in chronic HIV-1 infection. DNA methylation plays a major role in T(reg) lineage commitment and immune homeostasis, which may be regulated by HIV. To investigate the mechanisms of aberrant methylation of the T(reg) marker FOXP3 in HIV-1 infection, we evaluated the expression pattern of methylation-related enzymes and its correlation to FOXP3 methylation.

Methods: FOXP3 promoter methylation in the colon mucosa and peripheral blood from HIV-infected patients and control subjects was measured using Pyrosequencing. Gene expression pattern of DNA methylation enzymes in the colon mucosa was investigated by Microarray and quantitative reverse transcriptase-polymerase chain reaction analysis in the same subjects.

Results: FOXP3 promoter was significantly (P ≤ 0.0001) demethylated in HIV-infected patients compared with control subjects in both tissues. Expression of DNA methyltransferase 1 (DNAMT1), DNA methyltransferase 1-associated protein 1(DMAP1), methyltransferase-like 7B (METTL7B), and methyltransferase-like 10 (METTL10) were significantly down regulated in HIV-infected patients compared with controls and had a significant positive correlation to FOXP3 promoter methylation.

Conclusions: We present evidence suggesting that altered methylation pattern of FOXP3 and accordingly higher T(reg) frequency in gut mucosa of HIV-infected patients may be because of aberrant methylation processing in HIV.

Conflict of interest statement

Potential conflicts of interest. The authors do not have a commercial or other association that might pose a conflict of interest.

Figures

FIGURE 1

FOXP3 promoter methylation levels in PBMCs and colon tissue. Panel A shows that FOXP3 has a significant lower average percent methylation in HIV infected patients compared to control subjects;. The mean percent methylation and standard deviation in each group was as follows: control Lymphocytes (37.5 ± 4.9), control colon tissue (36.7 ±1.6), patients' lymphocytes (1.9 ± 0.6) and patients' colon tissue (1.2 ± 0.4). The difference in methylation was statistically significant between patients and control lymphocytes and colon tissue respectively (p< 0.0001, p< 0.0001 after adjustment of age, gender and race). In panel B the levels of FOXP3 methylation at different CpG islands were comparable in PBMCs and colon tissue from the same subject but different between controls and HIV-infected patients.

FIGURE 2

The level of FOXP3 gene expression in colon tissue examined by quantitative rt-PCR. Significantly (p= 0.009) higher relative expression levels of the FOXP3 gene (normalized to the housekeeping gene GAPDH) in patient samples compared to controls after adjusting for age, gender and race of patients) is shown.

FIGURE 3

Immunohistochemical staining of FOXP3 protein in colon tissue. Two representative slides of FOXP3 protein expression in colon tissue using Immunohistochemical staining as described in methods were shown. A and B represent the staining of FOXP3 in control while C and D represent the staining of FOXP3 in HIV patient colon tissue at 10X and 40X respectively. The mean score ± SD of FOXP3+ Treg cells were 20 ± 1.7 cells/HPF in HIV and 2 ± 0.85 cells/HPF in controls.

FIGURE 4

Relative expression levels of methylation enzymes using quantitative RT-PCR and correlation with FOXP3 methylation. In panel A, DNMT1; DMAP1, METTL7B, and METTL10 relative expression levels (normalized to the housekeeping gene GAPDH) were significantly (p≤ 0.05) down regulated in HIV infected patients compared to controls. In panel B, FOXP3 promoter methylation levels has a significant positive correlation to the relative expression levels of DNMT1, DMAP1, METTL7B, and METTL10 (Spearman r=0.7112, p= 0.0254, r = 0.8909, r = 0.8667, p= 0.0022 p=0.0011; and r= 0.7455, p= 0.0174 respectively).