Abdel-Hameed, Enass A. MD, PhD*; Ji, Hong PhD†; Sherman, Kenneth E. MD, PhD*; Shata, Mohamed T. M. MD, PhD*
Epigenetic modification of DNA leads to heritable changes in gene expression that are not coded by DNA sequence. The most commonly studied epigenetic changes include DNA methylation and chromatin modification. The importance of epigenetic changes such as the methylation of 5′-cytosine located in the CG-rich regions (islands) of DNA has emerged in cancer, autoimmune, and inflammatory diseases.1,2 The CpG islands are defined as short (0.2–2 kb) DNA regions with highly enriched cytosine/guanine dinucleotide.3 These CG-rich regions are usually located with higher frequency at the 5′ end of genes and are associated with transcriptional promoters.4 Most promoter CpG islands are nonmethylated or methylated at low levels regardless of transcription status, although high methylation of promoter CpG islands is commonly associated with repression of their transcription.1
Epigenetic modifications can modulate HIV-1 integration, transcription, and latency of infection. Integration of HIV-1 into the host genome is not likely to occur in transcriptionally inactive “methylated” regions of the genome.5 In latently infected CD4+ T cells, the transcription of integrated HIV-1 was suppressed by methylation of its promoter.6 Epigenetic modification of host genes can presumably affect HIV-1 transcription and replication. Furthermore, HIV-1 can induce de novo methylation of the host T-cell–specific genes through the induction of DNA methyltransferase 1 (DNMT1) in vitro.7 Recent data suggested that methylation of 5′-LTR of HIV is associated with the control of viral replication in a subset of patients characterized as long-term nonprogressor and elite controllers.8
The gastrointestinal (GI) tract houses most of the body's lymphocytes, and the GI mucosa represents a key compartment for HIV replication.9 DNA methylation regulates lineage commitment of lymphocyte subset T regulatory cell (Treg),10 which has an essential role in gut mucosal immune tolerance.11 Treg is CD4-positive (CD4+) T cells that inhibit immunopathology or autoimmune disease in vivo. The function of Treg depends on the expression of the transcription factor FOXP3 (forkhead box P3), which is considered the master switch for Treg. There are 3 conserved regions for methylation of FOXP3 in Treg: FOXP3 promoter, TGFβ-sensor, and TSDR-enhancer regions, which are differentially methylated in different subsets of T cells.10,12 FOXP3 promoter and TGFβ sensor are mainly demethylated in stable and induced Treg, respectively. Tregs inhibit immune responses by restraining excessive effector T-cell responses. Accumulated data suggested that there is an increased frequency of Treg among CD4+ T cells in GI mucosal tissue in simian immunodeficiency virus and HIV-1 infection.13 This increase in frequency of mucosal Treg was specifically found in HIV-1 infection but not in other viral infection such as Norovirus.14 However, the role of Treg in HIV infection is still controversial. Increased Treg frequency is associated with limited immune activation in HIV-exposed uninfected neonates and adults,15,16 and in ART treated patients,17 which has a beneficial effect to the host. On the other hand, Treg might exacerbate HIV infection by down regulation of specific immune responses toward the virus.18
The present study was designed to examine how HIV-1 infection modifies methylation of the genome, particularly in immune-related genes by which the virus can evade the host immune system, its association with clinical outcomes and possible underlying mechanisms. Specifically, we measured the levels of DNA methylation within FOXP3 promoter (as a biomarker for Treg) in peripheral blood mononuclear cells (PBMCs) and colon mucosa and studied how HIV-1 infection alters epigenetic modification of FOXP3. We also investigated the effect of aberrant DNA methylation level on FOXP3 gene and protein expression. In addition, we examined the relationship between FOXP3 methylation and clinical profile of HIV-1–infected patients and its correlation with immunological and virological status. Furthermore, we evaluated the expression pattern of methylation-related enzymes in the colon mucosa and its correlation to FOXP3 methylation.
Patients and Methods
All participants were recruited from University of Cincinnati clinics. Thirty milliliters of blood and 3 colonic mucosal biopsies 1–3 mm in size from the distal colon (30–45 cm from the anal verge) were obtained from patients and controls using flexible sigmoidoscopy according to the standard procedure. Consent forms were obtained from participating subjects according to a protocol approved by the University of Cincinnati School of Medicine Human Studies Committee and Institutional Review Board. To study the effect of HIV-1 infection on FOXP3 promoter methylation, 10 noninfected controls, and 10 HIV-1–infected subjects were enrolled in the study. All HIV-infected patients were receiving antiretroviral treatment for a median of 11 years. Half of the HIV-1–infected patients were coinfected with hepatitis C virus (HCV). Only 2 subjects were receiving anti-HCV treatment at the time of sample collection. Demographic data and characterization of the enrolled subjects are summarized in Table 1. Viral loads were determined in patient's plasma using COBAS AmpliPrep/COBAS taqMan HIV-1 Test v2.0 (Roche Diagnostics, Indianapolis, IN) with a threshold of 40 copies per milliliter. PBMCs were isolated from the blood by density gradient using Ficoll–Paque Plus, Histopaque (Sigma, St. Louis, MO) according to manufacturer's instruction. PBMCs were washed twice in RPMI-1640 (Gibco, Carlsbad, CA), counted and used immediately or stored in 10% dimethyl sulfoxide and 90% fetal bovine sera (Gibco) at −80°C for further analysis.
DNA Isolation and Na Bisulfite Modification
Tissue biopsies saved in RNA later were disrupted, and the lysate was homogenized in the appropriate volume of Buffer RLT Plus (Qiagen, Valencia, CA). The lysate was then centrifuged at maximum speed, and the supernatant was collected and used for isolation of DNA using AllPrep DNA/RNA Mini Kit (Qiagen) and treated with sodium bisulfite EZ DNA methylation direct kit (Zymo research, Irvine, CA) to convert unmethylated cytosine into uracil, although methylated cytosine remained unchanged.19 Bisulfite-modified DNA was amplified using polymerase chain reaction (PCR) primers for a specific CpG islands in FOXP3 promoter area (Qiagen). The SssI-treated human genomic DNA was used as 100% methylation control, and human genomic DNA amplified by GenomePlex-Complete Whole Genome Amplification Kit (Sigma) was used as the nonmethylated DNA control.
The PCR product was measured by quantitative Pyrosequencing using PyroMark Q96 MD (Qiagen) in the Pyrosequencing core Lab for Genomic and Epigenomic research (Division of Asthma Research, Cincinnati Children's Hospital Medical Center). Data were determined using the pyro Q-CpG methylation software (Qiagen) and were presented as percent methylation of each CpG dinucleotide tested.
Gene expression pattern was investigated by Chip Selection Human Gene 1.0 ST microarray according to manufacturer's instructions. For detailed description, please see the Supplemental Methods (http://links.lww.com/QAI/A442).
Quantitative Reverse Transcriptase-Polymerase Chain Reaction and Gene Expression Analysis
RNA was converted to cDNA using QuantiTect reverse transcription kit (Qiagen). The prepared cDNA was amplified using QuantiTect primer assay for FOXP3 (NM_014009), METTL10 (NM_212554), METTL7 (NM_152637), DMAP1 (NM_019100), DNMT1 (NM_001379), and GAPDH (NM_002046) and the QuantiTect SYBER Green PCR kit (Qiagen) according to the manufacturer's instructions.
FOXP3 protein expression was evaluated by Immunohistochemistry. For detailed description of procedure and analysis, please see the Supplemental Methods (http://links.lww.com/QAI/A442).20,21
The Student t test was used to examine differences between groups with a significance value at P ≤ 0.05. Correlations between parameters measured were calculated using Spearman correlation coefficient for patients and controls.
For promoter methylation, the average percent methylation of the selected CpG sites was compared between the healthy controls and the HIV-1 infected subjects. The initial screening experiment revealed that CpG sites tested in the FOXP3 promoter area have a significant lower average percent methylation in HIV-1 infected patients compared with the controls. The mean percent methylation and SD in each group were as follows: control lymphocytes (39.6 ± 1.25), control colon tissue (35.4 ± 2.8), HIV lymphocytes (1.9 ± 0.1), and HIV colon tissue (1.8 ± 0.4). No significant difference was seen between HIV and HIV/HCV co-infected patients; therefore, in later analysis, we grouped them together. The difference in methylation was statistically significant between patients and controls in both lymphocytes and colon tissue, respectively (P < 0.0001 and P < 0.0001 adjusted for age, gender and race) (Fig. 1A).
The methylation status of FOXP3 was compared in PBMCs and colon tissue of 5 HIV-1–infected patients and 6 control subjects to determine if this altered methylation status is different between the 2 types of tissues within the same control and HIV-infected subjects. The level of FOXP3 methylation was comparable in PBMCs and colon tissue from the same subject as shown in the heat map methylation profile (Fig. 1B).
To study the effect of FOXP3 promoter methylation on its gene expression in colon tissue, FOXP3 gene expression was quantified by reverse transcriptase-polymerase chain reaction (RT-PCR). As shown in Figure 2, there was significantly (P = 0.009) higher expression level of the FOXP3 gene in HIV-infected patient samples compared with controls. The methylation of FOXP3 promoter was significantly negatively correlated with the relative expression of the gene in colon tissue (Spearman r = −0.6606, P = 0.0438). Immunohistochemistry of colon tissues revealed a higher FOXP3 protein expression level and a higher frequency of infiltrating FOXP3+ Treg in colon tissue from HIV-1–infected patients compared with the control [mean score ± SD were 20 ± 1.7 cells per high-power field (HPF) and 2 ± 0.85 cells per HPF, respectively; Fig. 3; see Figure S1, Supplemental Digital Content, http://links.lww.com/QAI/A442].
All HIV-infected patients included in this study were receiving antiretroviral treatment with successful control of viral load except for 1 patient (1074 copies/mL) (Table 1).
The CD4+ count in HIV-1–infected patients ranged from 320 to 581 cells per microliter with an average ± SD = 472 ± 99. In contrast, in the noninfected controls, the range was 420–890 cells per microliter and the average ± SD was 699 ± 138. The overall difference in CD4+ count was significant between the control and patient groups (P < 0.000) (Table 1). The level of methylation of FOXP3 promoter is significantly positively correlated with CD4+ count (Spearman r = 0.5963, P = 0.0055).
In humans, DNA methylation is maintained by DNA methyltransferases and modified by other methylation enzymes. It has been previously shown that HIV-1 can induce de novo methylation of the host T-cell–specific genes through the induction of DNA methyltransferase 1 (DNMT1) in vitro.7 To identify possible mechanisms by which HIV infection modifies DNA methylation of host genes in vivo, we examined correlation between FOXP3 promoter methylation in HIV-1–infected patients and controls with the expression levels of DNA methyltransferase enzymes in colon tissue first by microarrays and further validated by quantitative RT-PCR. DNA methyltransferase 1–associated protein 1(DMAP1), methyltransferase-like 7B (METTL7B), and methyltransferase-like 10 (METTL10) was significantly down regulated in HIV-1–infected patients compared with the controls in both microarray analysis and quantitative RT-PCR measurement (P < 0.05) (Fig. 4A). In addition, we also found that DNMT1, which was not included in our array analysis, was significantly down regulated by quantitative RT-PCR (Fig. 4A). There was a significant positive correlation between the methylation level of FOXP3 and the expression levels of DNMT1, DMAP1, METTL7B, and METTL10 (Spearman r = 0.7112, P = 0.0254, r = 0.8909, P = 0.0022, r = 0.8667, P = 0.0011; and r = 0.7455, P = 0.0174, respectively) (Fig. 4B).
HIV infection is accompanied by a perturbed GI mucosal immune response with subsequent persistent inflammation and viral propagation.9 Viruses have developed several mechanisms by which they can escape the host immune response. Aberrant methylation of the HIV-1 and its surrounding environment is 1 mechanism that might help the virus escape the host immune response and maintain its existence.22 Decreased methylation of the FOXP3 promoter can lead to the induction and stabilization of Treg, a key player in maintaining immune homeostasis of the gut mucosa.23 However, the effect of HIV-1 infection on the epigenetic modification of the FOXP3 promoter, a marker of Treg cells, is unknown.
The present study was designed to examine the effect of HIV-1 infection on Treg by testing DNA methylation level of FOXP3 and its expression in colon mucosa and PBMCs from HIV-1–infected subjects. Our results indicate that the tested FOXP3 promoter area had a significantly lower level of methylation in colon mucosa and PBMCs from chronic HIV-1–infected patients as compared with control subjects (Fig. 1). This lower level of methylation was significantly associated with higher number of Treg infiltrating the colon mucosa of chronic HIV-infected patients and is in agreement with earlier reports that showed an increased number of infiltrating Treg in the gut mucosa of HIV-1–infected patients despite lower CD4+ counts24 (Fig. 3 and Table 1). However, the underlying mechanism of Treg induction in the colon mucosa of HIV-infected subjects was not clear. There is strong evidence suggesting that FOXP3 expression is regulated by DNA methylation of conserved elements in its promoter.12 Others have also suggested that decreased methylation of FOXP3 promoter might be considered a marker of higher percentage of natural Treg.25 At the cellular level, CD4+CD25hi FOXP3+ T cells (Treg) were not methylated, although CD4+CD25lo (activated T cells) displayed intermediate FOXP3 methylation.26 Additionally, FOXP3 expressing cells exhibited suppressive abilities that correlate to the methylation status of the FOXP3 promoter.26 Moreover, the use of demethylation agents, such as 5 aza-2′ deoxycytidine leads to increased expression of FOXP3 in natural killer cells.27 Collectively, our data suggested that decreased methylation of FOXP3 promoter of mucosal T-cell populations is the cause of increased FOXP3 expression and consequently increased Treg frequency in chronic HIV infection.
We next tested the difference in FOXP3 methylation in 2 tissues (PBMCs and colon tissue) obtained from the same individual assuming that there will be an intratissue methylation difference as reported earlier.3 No difference in the methylation level of FOXP3 promoter region in colon tissue or PBMCs either in control or HIV-infected subjects were observed. This suggests that the lower level of methylation in FOXP3 promoter is a systemic effect of HIV infection. It also indicates that the region we investigated is not a tissue-specific differentially methylated region; instead, methylation level in this region may serve as a cross-tissue uniform biomarker for HIV infection. Several mechanisms have been suggested to explain the increased frequency of Treg during the course of HIV infection.28 Increased proliferation and expansion of Treg population in the gut was documented earlier. However, this proportional Treg expansion was controlled by antiretroviral treatment.29 In this study, all patients were receiving antiretroviral treatment and their viral load was controlled, except one, which suggests that Treg expansion does not play a role in the methylation pattern observed in the HIV patients examined. Another possibility is the increased homing of FOXP3+ Tregs from blood to the lymphoid tissue in the gut,30 which is associated with slower restoration of CD4+ T in the gut compared with the blood in treated HIV-infected patients.31 This dynamic change in T-cell homing might lead to apparent increase in gut Treg. However, we noticed the same level of FOXP3 demethylation in both the PBMCs and colon tissue in the treated HIV-infected patients examined in our study.
Although the presence of HIV/HCV coinfection further impairs liver function and augments the severity of microbial translocation and immunopathology in coinfected patients,32 in our study, HCV coinfection did not further modify the level of methylation or expression of FOXP3 in the colon tissue, which suggests that the effect on Treg is mainly because of HIV. Because HIV infection alone already decreased the level of FOXP3 methylation to nearly zero, furthermore HCV infection may use mechanisms other than FOXP3 promoter methylation to exert its negative impact in patients. We noticed a higher methylation level for IL-17 promoter in the same HIV-infected patient samples (see Figure S2, Supplemental Digital Content, http://links.lww.com/QAI/A442) compared with FOXP3 promoter (manuscript in preparation). In addition, in contrast to FOXP3 promoter, there is no significant difference for IL-17 promoter methylation between patients and controls in PBMCs, although a significant (P = 0.01) higher methylation of IL-17 promoter was noticed in HIV colonic tissue. This differential methylation of genes in T cells indicates that the lower methylation in FOXP3 promoter is a unique effect of HIV infection on Treg cells master-regulator gene.
It is not clear if FOXP3 expression affects HIV replication in chronic HIV-infected patients. However, in an in vitro model of HIV infection, it has been shown that FOXP3 expression increases HIV-1 gene expression through histone acetylation of HIV long terminal repeat. The opposite effect was found on the IL-2 promoter.32 Lower methylation of the HIV long terminal repeat was also reported in latently infected resting CD4+ T cells isolated from HIV-1–infected individuals receiving antiretroviral therapy.33 However, in that study, the methylation pattern of subsets of T-cell promoters were not examined.
Whether HIV infection has a direct or indirect effect on the methylation of FOXP3 is a matter of a debate and needs further investigation. Although the development of in vitro model with Treg cells transduced with HIV infectious clones might seem as a reasonable approach to investigate this further, this model has some limitations; including the lack of sufficient numbers of Treg which would be resistant to apoptosis by replicating HIV virus,34 and the inability to create a microenvironment that represents Treg cells in vivo during chronic HIV infection.
To investigate whether the level of FOXP3 promoter methylation is influenced by immunological status in HIV-infected patients, the association between FOXP3 promoter methylation level and CD4+ counts was analyzed. CD4+ T-lymphocytes are the primary target for HIV infection and CD4+ count is a useful biomarker to monitor disease progression and responsiveness to antiretroviral therapy. In this study, all the HIV-infected patients were receiving antiretroviral treatment for a minimum of 3 years and improved their CD4+ counts but not to the normal level. Interestingly, we found a significant positive correlation between the level of FOXP3 methylation and blood CD4+ counts. Decreased levels of FOXP3 methylation is associated with an increase in FOXP3+ Treg number in the colon mucosa and correlated with lower CD4+ count in patients. This can be explained by previous observations that activated Treg can nonspecifically suppress HIV-specific inflammation and recruitment of CD4+ to the colon mucosa.35 Almost all HIV-infected patients included in this study had a controlled viral replication by a successful antiretroviral therapy, which limits our ability to study the correlation between FOXP3 methylation and viral loads.
To investigate possible mechanisms of aberrant methylation of FOXP3 by HIV-1 infection, gene expression pattern of DNA methylation enzymes in the colon mucosa from chronic HIV-infected patients and control subjects was tested by Microarray analysis and quantitative RT-PCR. Expression of DNMT1, DMAP1, METTL7B, and METTL10 were significantly down regulated in HIV-infected patients compared with controls and had a significant positive correlation to FOXP3 promoter methylation (Fig. 4). DNA methyltransferases are a group of enzymes that catalyze the transfer of the methyl group from the ubiquitous methyl donor S-adenosyle-L-methyonine (AdoMet) to different accepting molecules, including proteins, small molecules, lipids, and nucleic acids.36 Both METTL10 and METTL7 protein has been annotated in UniProt database as having a 7 beta-strand AdoMet binding domain.37 To our knowledge, the association between the expression pattern of these 2 enzymes and FOXP3 methylation was reported for the first time by our study and has not been reported previously in HIV infection. The specific methylation reaction catalyzed by METTL10 and METTL7B remains to be characterized.
In contrast, DMAP1 protein is known to be a corepressor of transcription that stimulates universal and local DNA methylation.38 Knocking down DAMP1 in human cell lines caused hypomethylation of the tumor suppressor gene p16 that leads to cell growth arrest. In a similar manner, the knocked down DMAP1 triggered hypomethylation of DNA repair products with resulting elevated genomic instability.39 The lower expression level of DMAP1 that we observed in the colon tissue from HIV-infected patients may indicate a low level of DMAP1 activity.
DNMT1 has been described as the main enzyme responsible for do novo methylation and propagation of DNA methylation pattern in vertebrates.40 In addition, DNMT1 was shown to maintain a repressive chromatin structure (deacetylated) through its binding to DMAP1 and recruitment of the enzyme histone deacetylase 2 (HDAC2) to the replication foci of late S phase in vivo.41 In agreement with previous report of lower methylation of HIV virus promoter in treated chronic HIV-infected patients,35 our data show lower expression of DNMT1 in chronic HIV-1–infected patients under treatment compared with controls. The lower expression of DNMT1 correlated positively with methylation status of FOXP3 promoter in patients and controls. Interestingly, although there is evidence that HIV-1 early proteins (Nef and Tat)–induced DNTM1 expression from a reporter construct transfected Hela cells,7 FOXP3 suppresses the transcriptional activity and the promoter DNA binding of AP-1,42 which was shown in the previous study to inhibit the induction of DNMT1 expression by HIV-1.7 Our data highlight for the first time the possible mechanisms by which HIV-1 infection may alter methylation pattern of FOXP3 in Treg and immune homeostasis. Limitations of our study, including that it is cross-sectional and did not have samples from HIV-infected untreated patients to investigate the effect of treatment.
In summary, we found lower level of FOXP3 promoter methylation in the gut mucosa and blood of chronic HIV-1–infected patients who were receiving prolonged antiretroviral therapy. This decreased methylation level of FOXP3 promoter was significantly associated with changes in the gene and protein expression of FOXP3 and a higher frequency of infiltrating Treg in the gut mucosa of infected patients. Collectively, our data support that higher Treg frequency in gut mucosa of HIV-infected patients may be caused by aberrant methylation process associated with HIV infection.
The authors thank the entire participants in this study particularly the patients. The authors thank the Pyrosequencing Lab for Genomic and Epigenomic Research, Digestive Health Center, The Gene Expression Microarray Core, and Bioinformatics Core in Cincinnati Children's Hospital Medical Center.
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