HIV infection of human regulatory T cells downregulates Foxp3 expression by increasing DNMT3b levels and DNA methylation in the FOXP3 gene
Pion, Marjorie; Jaramillo-Ruiz, Didiana; Martínez, Alberto; Muñoz-Fernández, M. Angeles; Correa-Rocha, Rafael
Laboratorio de Inmunobiología Molecular, Instituto de Investigación Sanitaria Gregorio Marañón, Madrid, Spain.
Correspondence to Rafael Correa Rocha, PhD, Laboratorio de Inmuno Biología Molecular, Instituto de Investigación Sanitaria Gregorio Marañón, Dr Esquerdo, 46, 28007 Madrid, Spain. Tel: +34 91 586 8565; fax: +34 91 586 8018; e-mail: firstname.lastname@example.org
Received 21 January, 2013
Revised 11 April, 2013
Accepted 17 April, 2013
Supplemental digital content is available for this article. Direct URL citations appear in the printed text and are provided in the HTML and PDF versions of this article on the journal's Website ( http://www.AIDSonline.com).
Regulatory T cells (Tregs) play an important role in infections modulating host immune responses and avoiding overreactive immunity. The mechanisms underlying their action in HIV-infected patients have not been well established. HIV can infect Treg, but little is known about the effects of the infection on Treg phenotype and function. The objective of this study was to investigate whether in-vitro HIV infection modifies the phenotype and suppressive capacity of Treg cells.
Because Treg cells are a subset of CD4+ T cells, HIV infection could produce alterations in the phenotype and methylation pattern of Treg disturbing the functionality of these cells.
Isolated Treg cells from healthy volunteers were cultured in the presence of HIV-1, and phenotype, methylation pattern of FOXP3 locus, cytokine secretion profile and suppressive function of infected Treg were analysed in comparison with noninfected Treg.
We demonstrate that HIV-1 directly infects Treg and deregulates the function and the phenotype that define these cells. HIV infection downregulates the Foxp3 expression in Treg, which is followed by the loss of suppressive capacity and alterations in cytokine secretion pattern, with decreased production of transforming growth factor-beta (TGF-β) and an increased production of interleukin (IL)-4. Foxp3 downregulation in HIV-infected Treg was related to an increase in the expression of DNA methyltransferase3b (DNMT3b) associated with higher methylation of CpG sites in the FOXP3 locus.
These findings are pivotal to our understanding of the role of Treg in HIV infection and indicate that regulatory function could be seriously impaired in HIV-infected patients contributing to the immune hyperactivation.
Regulatory T cells (Tregs) are a specialized subpopulation of CD4+ T cells that constitutes a crucial cellular component of a normal immune system and plays a pivotal role in establishing and maintaining self-tolerance and immune homeostasis . Tregs exert a regulatory effect on immune cells by suppressing the proliferation and function of diverse immune subsets . Consequently, Tregs play a fundamental role in human diseases such as allergy, autoimmune diseases, cancer and infections [3–5].
Most infections induce (or recruit and expand) Treg to modulate host immune responses and thus avoid overreactive immunity . Immune hyperactivation associated with HIV infection could lead to erosion, depletion and exhaustion of the CD4+ T-cell pool compromising the specific immune responses against HIV. By that, hyperimmune activation is considered a reliable predictor of AIDS progression . The role of Treg in HIV infection is critical because of their implication preventing hyperactivation of the immune system . However, the findings associated with Treg from HIV-infected patients are not clear, and several groups have shown that Treg numbers are either decreased [9–11] or increased [12–14] in HIV infection.
As Tregs express CD4, chemokine (C-X-C motif) receptor 4 (CXCR4) and chemokine (C-C motif) receptor 5 (CCR5), they are also susceptible to being infected by both R5-tropic and X4-tropic HIV [10,15]. The effects of HIV infection in conventional CD4+ T cells are numerous, but little is known about the potential effects of HIV infection on the phenotype and function of Treg, which is essential to understand the role of Treg in HIV-infected patients.
Foxp3 is the major transcription factor determining the fate and identity of Treg and it is essential in the maintenance of suppressive function . The principal difficulty when defining the Treg functional population is that Foxp3 can also be induced in peripheral naive CD4+CD25− T cells , but it confers neither stable expression of Foxp3  nor suppressive activity [19,20]. Several groups have demonstrated that epigenetic regulation is crucial for controlling the expression of the FOXP3 locus . A demethylated pattern in FOXP3 promoter and other proximal regulatory elements is a prerequisite for stable Foxp3 expression and the subsequent suppressive phenotype in Treg [21,22]. Methylation status of Foxp3 could be disturbed by HIV infection because methylation is a mechanism by which the cell genome is protected against expression of foreign DNA such as viruses . During retroviral infection, methylation is increased throughout the viral genome, particularly at the viral long terminal repeats (LTRs), providing a mechanism of suppression of viral expression and latency for HIV [24,25]. In fact, HIV infection increases the expression and activity of DNA methyltransferases (DNMTs), which are the enzymes responsible for DNA methylation [26,27].
Our objective was to determine whether direct infection of Treg with HIV could lead to their phenotypical or functional deregulation. We performed an extensive study in which we infected in-vitro isolated Treg from healthy donors with HIV. We report for the first time that HIV infection of Treg modifies its phenotype and functionality in a process mediated by methylation mechanisms. We propose that the homeostatic and suppressor effect of Treg during HIV infection is generally lost once infection is established. This additional piece of the jigsaw could increase our understanding of the general deregulation and hyperactivation of the immune system during HIV infection.
Materials and methods
Peripheral blood samples were obtained from 15 healthy adult volunteers (25–40 years old) after informed consent in accordance with local ethical committee approval. Blood was immediately processed after extraction in the Spanish HIV BioBank  and peripheral blood mononuclear cells (PBMCs) were purified using Ficoll-Paque according to manufacturer's protocol.
Purity and viability of cultured Treg cells
Untouched CD4+ T cells isolated with magnetic beads (Miltenyi Biotec, Bergisch Gladbach, Germany) were stained and CD4+CD25+CD127low Treg cells were isolated using an ASTRIOS cell sorter (Beckman Coulter, Barcelona, Spain) (suppl. Figure S1A, B, http://links.lww.com/QAD/A353). Flow cytometry analysis of sorted Treg indicates that in all experiments, more than 95% of sorted cells were CD4+ without contaminating CD8+ cells (Fig. S1C, D, http://links.lww.com/QAD/A353) even after culture (Fig. S2A, http://links.lww.com/QAD/A353); more than 90% were CD4+CD25+CD127-, from which more than 85% were Foxp3+ (Fig. S1B, E, http://links.lww.com/QAD/A353). Viability of infected and noninfected Tregs was determined at all the time points using the Fixable Viability Dye (eBiosciences, San Diego, California, USA), which is compatible with Foxp3 staining in permeabilized cells. Results indicate that viability of both noninfected and HIV-infected cells remains above 70% until 5 days postinfection and decreases remarkably after 10 days of culture (day 7 postinfection) notably in HIV-infected Treg (Fig. S2B, http://links.lww.com/QAD/A353).
Virus stock production
Virus stock HIVNL4–3 was produced by infection of MT2 cells with NL4–3 virus stock coming from previous transient transfection of pNL4–3 in 293T cells. Virus stock of R5 HIV-1NL(AD8)–tropic strain was produced by transient transfection of pNL(AD8) in 293T cell lines [pNL4–3 and pNL(AD8) plasmids are from NIH AIDS Research Program]. Viral titres were expressed as infectious units per millilitre and infection was done through multiplicity of infection (MOI), where MOI of 1 corresponds to 1 IU per one cell.
Cell culture and HIV infection
Freshly sorted Tregs were activated with Dynabeads Human Treg Expander (Invitrogen, Paisley, UK) following manufacturer's recommendations. Treg cells were then infected with HIVNL4–3 or HIVNL(AD8) (MOI from 0.01 to 1) for 3 h; cells were then extensively washed and cultured in the presence of 500 U/ml of interleukin (IL)-2. Noninfected Treg cells and CD4+CD25− non-Treg cells from the same donor were processed in parallel and used as controls. Controls for inhibition of viral replication were performed with zidovuline (ZDV) (Retrovir; GlaxoSmithKline, Brentford, UK) and T-20 (Genentech, San Francisco, California, USA).
Real-time PCR amplification
RNA and genomic DNA were isolated by RNA/DNA micro extraction kit (Qiagen, Madrid, Spain). RNA integrity was analysed with Agilent 2100 bioanalyzer (Agilent Technologies, Waldbronn, Germany) and samples with degraded RNA were ruled out of the analysis. Real-time PCR was performed with Brilliant II QPCR Master Mix (Agilent Technologies). The primers used to amplify mRNA are listed in Table S1A, http://links.lww.com/QAD/A353. Gene mRNA levels detected by real-time (RT)-PCR were normalized with glyceraldehyde-3-phosphate dehydrogenase and hypoxanthine guanine phosphoribosyl transferase housekeeping genes.
Cultured Tregs were washed and stained with antihCD4-ECD, antihCD25-PECy5 and antihCD3-PECy7 (Beckman Coulter). Fixation and permeabilization for intracellular staining was done with Anti-Human Foxp3 Staining Set (eBiosciences), and cells were stained with anti-Foxp3-PE (eBiosciences) and antip24 protein (KC57-FITC; Beckman Coulter). Data acquisition was performed in a Beckman Coulter GALLIOS cytometer.
Methylation analysis of FOXP3 gene
Only male donors were used in order to avoid possible artefacts due to random X chromosome inactivation in females that could affect the methylation analysis of FOXP3 gene (localized in chromosome X). Analysis was performed at day 7 postinfection, when highest modulation by HIV was reported . Genomic DNA from cultured Treg cells was bisulphite converted by EpiTect Bisulfite Kit (Qiagen) according to the manufacturer's instruction. Three specific CpG-rich regions were amplified by PCR using primers for specific targets in bisulphited DNA (Table S1, http://links.lww.com/QAD/A353) and PCR products were cloned into a pCR4-TOPO vector using a TOPO TA Cloning Kit (Invitrogen). Twenty individual clones from each region and from each sample were isolated and sequenced.
Treg-suppressive effect on T-cell proliferation and activation
Functional analysis was performed at day 5 postinfection when rates of Treg viability were still high. We analysed the Treg-suppressive effect on T-cell proliferation. Freshly purified allogenic naive T cells (Teff) were purified from buffy coat and stained with CFSE (5-(and -6)- carboxyfluorescein diacetate succinimidyl ester) (Invitrogen) following manufacturer's recommendations. Allogenic antigen-presenting cells (APCs) were purified from buffy coat by magnetic beads as a negative fraction of CD3+ T cells from PBMC and incubated with 20 μg/ml of Mitomycin C (Roche Applied Biosystems, Barcelona, Spain) for 3 h at 37°C. T-cell proliferation assay was performed mixing Teff, APC (to promote Teff proliferation) and different ratios of infected or noninfected Treg. Cell cultures were then stimulated with CD3 Dynabeads, and after 5 days, CFSE signal of lymphocytes was analysed by flow cytometry. In addition, the capacity of Treg to suppress T-cell activation was determined by measuring the percentage of CD69 and CD154 in activated PBMCs cocultured with noninfected and HIV-infected Treg. This analysis was performed using the Regulatory T-Cell Function Kit (Beckton-Dickinson, Franklin Lakes, New Jersey, USA) according to manufacturer's protocol.
p24gag and cytokine production
Viral entry and infection was confirmed by measuring the concentration of p24gag in the culture supernatant by ELISA (Innotest HIV-1 antigen mAb; Innogenetics, Gent, Belgium). Moreover, cytokine levels were quantified in the supernatant of cell cultures at day 7 postinfection using Human Th1/Th2/Th9/Th17/Th22 13plex Kit and Human TGF-β1 Flow Cytomix Kit (eBioscience).
Statistical analysis was performed using statistical software SPSS (SPSS Inc., Chicago, Illinois, USA). Nonparametric Mann–Whitney and Wilcoxon paired tests were used. Correlation between variables was established by Pearson's correlation test. A P value of less than 0.05 by two-sided test was considered significant.
HIV infection downregulates Foxp3 expression in Treg
Freshly sorted Tregs were infected with CXCR4-tropic (X4) HIV-1 at three different MOIs for 3 h. High levels of HIV replication were detected 3 days postinfection by p24gag production in the supernatant (Fig. 1a) and by intracellular detection of p24gag protein (KC57) (Fig. 1b). After 3 days of infection, we observed that expression of CD4 in HIV-infected Foxp3+ cells decreased in all the donors (Fig. S2C, http://links.lww.com/QAD/A353). However, the percentages of CD3+T cells were comparable between noninfected Treg (NI-Treg) and HIV-infected Treg (Fig. S2D, http://links.lww.com/QAD/A353). Therefore, because CD4 surface expression could be affected by the infection, gating strategy for Treg cells detection in this study consisted first in identification of the living cells (using fixable viability dye), then in gating CD3+ cells and finally in analysing the percentage of CD25+Foxp3+ cells.
Interestingly, infection of Treg with HIV-1 led to a decrease in Foxp3 expression. We observed that HIV-infected Treg with a higher expression of intracellular p24gag (KC57) showed a lower Foxp3 mean of fluorescence intensity (MFI) than cells with lower p24gag expression (Fig. 1c), and infected cells with preserved Foxp3 expression were negative for KC57 (Fig. S3, http://links.lww.com/QAD/A353). Foxp3 downregulation was observed after as few as 3 days of infection and the decrease was especially marked after 7 days (Fig. 1d). Foxp3 decrease in comparison with NI-Treg was significant at MOI equal to 0.1 after 3 days of infection (n = 10, P = 0.005) and at day 5 (n = 6, P = 0.028), whereas at day 7 (n = 10), the decrease was significant for both MOI tested: 0.01 (P = 0.018) and 0.1 (P = 0.003) (Fig. 1d). Downregulation of Foxp3 in HIV-infected Treg was confirmed by determining Foxp3 mRNA level using real-time PCR, and the results also demonstrated a significant reduction in Foxp3 expression (P = 0.018 for the MOI of 0.1) (Fig. 1e).
We also investigated whether infection with CCR5-tropic (R5) strains at MOI equal to 0.1 produced the same effect on Foxp3 expression. We confirmed that R5 viruses infect Treg as previously described , but levels of p24 (KC57) produced by R5-infected Treg (Fig. S4A, http://links.lww.com/QAD/A353) and frequency of KC57+ cells (Fig. S4B, http://links.lww.com/QAD/A353) were lower than by X4-infected Treg. We observed that after 5 days of infection, R5-tropic virus had no effect in Foxp3 expression when compared with NI-Treg (Fig. 1f, Fig. S5A, http://links.lww.com/QAD/A353). Moreover, regarding R5-infected Treg with high expression of KC57, they did not show differences in the expression of Foxp3 in comparison to R5-infected Treg showing lower expression of KC57 (Fig. S5B, http://links.lww.com/QAD/A353). To confirm that productive infection is necessary for the observed Foxp3 downregulation, we treated Treg with 5 μmol/l of ZDV (n = 5) or 20 μmol/l of T-20 (n = 3) before infection with X4 virus. As expected, ZDV or T-20 treatment prevented Treg infection (Fig. S4B, http://links.lww.com/QAD/A353) and treated cells showed frequencies of Foxp3 comparable to NI-Treg (P > 0.05) (Fig. 1f).
Summing up, our data indicated that HIV-infected Treg cells experienced phenotypic changes determined notably by a marked downregulation of Foxp3 expression. This downregulation was observed with X4-tropic but not with R5 virus and was more intense in HIV-infected Treg with a higher level of viral replication.
Methylation of FOXP3 gene is increased in HIV-infected Treg
Because stable expression of Foxp3 in Treg requires epigenetic regulation based on DNA methylation, we investigated whether the observed decrease in Foxp3 after HIV infection could be mediated by changes in the methylation pattern of the FOXP3 gene regulatory sites. We analysed an upstream enhancer region  and two regions located in FOXP3 gene (FOXP3 promoter, Foxp3 conserved noncoding sequence 2 (CNS2) [30,31]) in noninfected and HIV (X4)-infected Treg obtained from five different male volunteers.
Average level of methylation in both noninfected and infected Treg was donor dependent. Figure 2a shows the mean increase in methylation detected in the HIV-infected Treg compared with NI-Treg. Interestingly, no changes were observed in methylation for the enhancer region, but HIV infection induced a significant increase in methylation in all the five experiments for both the promoter (3.3-fold; P = 0.043) and the CNS2 region (3.8-fold; P = 0.043) (Fig. 2a). Looking at individual CpG sites, HIV infection did not preferentially induced methylation in particular CpG positions (Fig. 2b). Therefore, our data demonstrate that infection of Treg with HIV induced a general increase in the CpG methylation pattern in two of the three regions that are pivotal for the regulation of Foxp3 expression in Treg cells.
DNA methyltransferase expression levels were altered in HIV-infected Treg
DNA methylation is performed by three known DNMTs (DNMT1, DNMT3a and DNMT3b), which are responsible for both maintenance and de-novo methylation. We measured the effect of X4-tropic HIV infection on the expression of DNMTs in Treg by real-time PCR. HIV infection did not significantly change the expression of DNMT1 and DNMT3a at the two MOIs tested (Fig. 3a,b). However, a marked increase was observed for DNMT3b expression in HIV-infected Treg when compared with NI-Treg (P < 0.05; increase of 7.3-fold and 11.3-fold for MOI of 0.01 and 0.1, respectively) (Fig. 3c). Expression levels of DNMT1 and DNMT3a mRNA in non-Treg CD4+ T cells were similar to those of noninfected or HIV-infected Treg. Nevertheless, DNMT3b in NI-Treg (which was seven-fold lower than in non-Treg cells) reached values comparable to those recorded in non-Treg CD4+ T cells when Tregs were infected (Fig. 3c).
HIV infection impairs the suppressive capacity of Treg
Foxp3 is a key factor for the suppressive function of Treg. Therefore, we analysed whether Foxp3 decrease caused by infection modified the suppressive capacity of Treg on both the proliferation and activation of T effector cells. First, we measured the proliferation of allogenic T effector cells (Teff) labelled with CFSE cocultured at different ratios with NI-Treg or HIV-infected Treg. The results showed that HIV-infected Tregs had a significantly impaired suppressive capacity compared with NI-Treg for the three ratios tested (Fig. 4a, P < 0.05). Moreover, the proliferation of Teff cocultured with HIV-infected Treg was equivalent to the proliferation of Teff alone, indicating a complete loss of suppressive function in HIV-infected Treg. As a control, we performed the same experiments using infected and noninfected CD4+ T cells instead of Treg, and the results demonstrate that presence of HIV or non-Treg cells in the cocultures did not modify the proliferation of Teff (Fig. S6, http://links.lww.com/QAD/A353).
We also investigated the suppressive effect of NI-Treg and HIV-infected Treg on the activation of allogenic PBMCs (Fig. 4b). We measured the frequency of activation markers (CD154 and CD69) in PBMCs stimulated by anti-CD3/CD28 coated beads cocultured with Treg (Treg: PBMC ratio 2 : 1). Suppressive capacity of Treg was calculated as the percentage of suppression of activation markers in stimulated PBMCs: 100 – [(%positive in presence of Tregs / %positive in absence of Tregs) × 100]. NI-Tregs were capable of suppressing both CD69 and CD154 expression in PBMCs by more than 25%. However, the suppressive capacity of HIV (X4)-infected Treg was less than 8% for both markers, and it was significantly lower than in NI-Treg (CD69: P = 0.035; CD154: P = 0.034). When HIV-infected Tregs were previously treated with 5 μmol/l of ZDV, we observed that the suppressive effect was preserved and ZDV-treated Treg inhibited the PBMC activation in more than 20%, which was comparable to the suppressive capacity of NI-Treg (CD69: P = 0.281; CD154: P = 0.339).
Summing up, our data showed that HIV-infected Treg lost their suppressive effect on the proliferation and activation of T effector cells when compared with NI-Treg, assuming that loss of Foxp3 was actually followed by the loss of function of these cells.
HIV infection modifies the cytokine secretion pattern of Treg
We monitored whether the loss of Foxp3 expression was associated with changes in the cytokine production of Treg. Production of cytokine measured in culture supernatants represented the accumulation of cytokines production over 7 days of culture. IL-12 (Th1), IL-5, IL-6 and IL-10 (Th2), and IL-17A (Th17) were undetectable in supernatant of both NI-Treg and HIV-infected Treg. We did not observe differences in the production of IL-9 (Th9), and although IL-13 (Th2) expression seemed to be lower in HIV-infected Treg, the difference with NI-Treg was not significant (P = 0.499; data not showed). However, HIV infection led to a significant increase (P = 0.018) in IL-4 production, and the production of transforming growth factor-beta (TGF-β), was significantly lower (P = 0.028) in HIV-infected Treg (Fig. 5). These data showed that HIV infection of Treg leads to a marked deregulation in the production of some cytokines that could influence the natural function of Treg.
The objective of this study was to determine how HIV infection affects the phenotype and properties of Treg. First, we confirmed that Treg cells are susceptible to being infected by HIV. This finding has been reported by other groups [10,15], clearly demonstrating that Treg cells are potential targets for HIV infection, although little is known about the effects of HIV infection on Treg. The first effect that we observed was a reduction in cell surface CD4+ expression in Treg that, as in other CD4+ T cells, could be mediated by the viral protein nef.
The main finding of this work is that in-vitro HIV(X4) infection induces a downregulation of Foxp3 in Treg cells. Although expression of Foxp3 is also partially lost in cultured noninfected Treg, probably due to the previously reported downregulation of Foxp3 in Treg upon repetitive stimulation and long-term culture , the decrease of Foxp3 after HIV infection was significantly greater in all experiments performed. Moreover, antiretroviral drugs such as ZDV and T20 were capable of reverting the effect of infection avoiding the loss of Foxp3 expression. Thus, a robust productive infection and not just exposure to HIV is crucial to disturb the Treg phenotype and function.
The lack of effect of R5 viruses on the Foxp3 expression is probably a consequence of the lower spread of these strains in Treg, as confirmed by the lower p24 production observed in R5-infected Treg. Tregs have been shown to be less susceptible to R5 viruses, and X4 viruses induced a more rapid infection in Treg . This fact would explain that after 5 days of infection with R5 viruses, we did not observe the same effects on Treg than with X4 viruses. Whether R5 viruses could have an effect after longer periods of incubation cannot be excluded, and further studies would be required to definitively resolve this important point.
Unlike another studies showing increased percentages of Foxp3+ cells in infected patients [12,14], our results point out that direct HIV infection of Treg induces a downregulation of Foxp3 expression. Other studies report that Treg frequencies are declined in peripheral blood of viremic patients but increased in lymphoid tissue wherein active viral replication occurs . Thereby, an increased proportion of Foxp3+ cells in HIV-infected patients could be explained by a transient induction of Foxp3 in non-Treg cells depending on the activation status or T-cell receptor stimulation (as demonstrated in HIV-infected patients ), or by the HIV-mediated depletion of non-Treg CD4+ T cells that would increase the relative proportion of Treg in the total CD4+ T-cell pool [12,35], but not by an increase of committed Treg. More recent articles demonstrate that, at a difference of the relative frequency, the absolute number of Foxp3+ T cells are clearly decreased in chronically HIV-infected patients [36–38]. The HIV-mediated downregulation of Foxp3 described here has not been previously reported and could constitute the molecular basis for the reduced absolute numbers of Foxp3+ cells described in HIV-infected patients and it explains why viremic patients have a lower frequency of Foxp3+ cells than aviremic patients .
Among the mechanisms that could mediate Foxp3 downregulation in HIV-infected Treg, epigenetic regulation is crucial for control of the FOXP3 locus expression and is a cellular mechanism altered by viral infections . Our results demonstrate that HIV infection increases the expression of DNMT3b in Treg reaching the levels observed in non-Treg CD4+ T cells, which are known to have a high methylation profile in the FOXP3 locus. These results correlate with previous studies showing that HIV infection increases the expression and activity of DNMTs when infecting CD4+ T cells [26,27,40]. Dnmt3b is specialized in inducing de-novo methylation of CpG sites in DNA, and binding sites for DNMT1 and DNMT3b have been detected in the FOXP3 locus . Then, the recruitment of these DNMTs could produce methylation of CpG residues and thus the repression of Foxp3. However, further studies must be performed to demonstrate definitively that DNMT3b increase is primarily responsible for the observed Foxp3 downregulation.
We hypothesize that HIV-1 infection by increasing Dnmt3b expression is responsible for the higher methylation in two of three analysed CpG regions associated with the FOXP3 locus. HIV-infected Treg showed an increased methylation into the promoter and CNS2 regions of the FOXP3 gene, which possess binding sites for different transcription factors [i.e. signal transducer and activator of transcription 5 (STAT5)], and have proven to be critical for the induction and stable maintenance of Foxp3 expression . We observed that these two regions were not totally demethylated in NI-Treg, which could be explained because partial methylation occurs in long-term cultures after activation . However, in all donors, HIV infection increased methylation in both regions, reaching mean values comparable to those reported by Miyara et al. for Foxp3lowCD45RA- or Foxp3- cells. Therefore, the degrees of methylation reached in HIV-infected Treg are not comparable to those of naive CD4+ T cells, but they could be high enough to inhibit Foxp3 expression and the suppressive function of these cells as shown previously .
DNA methylation of FOXP3 locus has been strictly associated with loss of suppressive activity [21,31]. Therefore, HIV-mediated methylation and consequent downregulation of Foxp3 expression would be responsible for the impaired suppressive capacity that we observed in HIV-infected Treg [18,31]. The loss of suppressive capacity in HIV-infected Treg was comparable to that reported in Foxp3low and Foxp3– cells, with a methylation profile similar to our values for HIV-infected Treg . It is interesting to note that a previous work described normal levels of suppression by HIV-exposed Tregs . These contradictory results could be explained by differences in the protocol used: Moreno-Fernandez et al. analysed the suppressive capacity after 3 days of infection with a R5-tropic virus, which did not inhibit the Foxp3 expression in our experiments; however, we observed the loss of suppressive capacity at day 5 postinfection with an X4-tropic virus. Our results are also in discrepancy with a study of Ji et al. showing increased suppressive capacity of HIV-exposed Treg. In that case, authors used resting Treg instead of activated Treg, in which both X4 and R5 HIV did not replicate (undetectable p24 after 5 days of HIV exposure) . As shown in our results, a productive infection and not just HIV exposure was necessary to produce the observed Foxp3 downregulation. In fact, in the presence of ZDV that prevents replication, the suppressive capacity of HIV-exposed Treg was not modified.
Downregulation of Foxp3 has also been associated with changes in the phenotype of Treg. It was shown that mature peripheral Treg develop into cytokine-producing effector T cells with a Th-cell phenotype if Foxp3 expression is attenuated . HIV-infected Treg showed reduced levels of TGF-β, which participates in the induction of Foxp3 expression and differentiation of induced Treg , and promotes natural Treg (nTreg) survival . Thus, TGF-β decrease observed in HIV-infected Treg could also jeopardize the survival of the nTreg population. In addition, our results demonstrate that HIV-infected Treg showed an increased production of IL-4, which is consistent with molecular findings showing that the IL-4 gene is directly suppressed by Foxp3 . A Th2 skewed cytokine pattern in Treg that have lost Foxp3 expression has also been previously reported  and could contribute to the reported switch from the Th1 to Th2 phenotype in HIV-infected patients . Interestingly, a similar switch to an increased production of Th2 cytokines by Foxp3+ cells that have lost their suppressive function has been recently described in HIV-infected patients .
In conclusion, our findings demonstrate that HIV infection of Treg leads to an alteration of the phenotype that defines these cells, as well as the cells’ suppressive capacity. Numerous factors, such as viral load, immune status and so on, could be implicated on the Treg alterations described in HIV-infected patients. However, our results provide a relevant piece of information to better understand the Treg dynamics and could provide the molecular basis for the reduced absolute numbers in HIV-infected patients reported by numerous studies. HIV infection of Treg would lead to a loss of Foxp3 expression and it could render Treg unable to suppress the immune responses. In the context of HIV infection, the loss of regulatory function could increase the generalized hyperactivation of the immune system, as reported in HIV-infected patients with low Treg frequencies , thus leading to a more rapid CD4+ T-cell decline and clinical progression to AIDS. The deleterious or beneficial effect of Treg in HIV infection warrants further study in vivo, but our findings pave the way for new therapeutic strategies to prevent HIV-mediated methylation and preserve the suppressive and homeostatic function of Treg in HIV-infected patients.
We thank the volunteers who participated in this study for providing blood samples. We thank R. Lorente and L. Díaz for technical assistance. We are grateful to Dr M. Miyara and Prof. S. Sakaguchi for providing us with the protocol for methylation analysis. We are grateful to Dr Moreno-Fernández and Dr Chougnet for providing us with the protocol for Treg cultures.
M.P. performed research, analysed data and contributed to the preparation of the manuscript; D.J. performed methylation and cytometry experiments and analysed data; A.M. performed methylation analysis and analysed data; M.A.M-F contributed to the design of the project and the preparation of the manuscript; R.C-R. was responsible for the overall study, designed the project and wrote the manuscript.
This work was supported by grants from Fondo de Investigación Sanitaria (FIS PS09/02618; FIS PI12/00934; FIS PS09/02523; FIS PI12/01763) and Red RIS (RD06/0006/0035; RD12/0017/0037). M. Pion is supported by the ‘Ramon y Cajal’ programme of Ministerio de Ciencia e Innovación (RYC-2009-05486). Rafael Correa-Rocha is supported by the Fondo de Investigación Sanitaria through the ‘Miguel Servet’ programme (CP07/00117). The funders had no role in study design, data collection and analysis, decision to publish or preparation of the manuscript.
Conflicts of interest
All the authors declare that they do not have a commercial or other association that might pose a conflict of interest.
1. Sakaguchi S, Yamaguchi T, Nomura T, Ono M. Regulatory T cells and immune tolerance. Cell. 2008; 133:775–787.
2. Tang Q, Bluestone JA. The Foxp3+ regulatory T cell: a jack of all trades, master of regulation. Nat Immunol. 2008; 9:239–244.
3. Cvetanovich GL, Hafler DA. Human regulatory T cells in autoimmune diseases. Curr Opin Immunol. 2010; 22:753–760.
4. Lund JM, Hsing L, Pham TT, Rudensky AY. Coordination of early protective immunity to viral infection by regulatory T cells. Science. 2008; 320:1220–1224.
5. Palomares O, Yaman G, Azkur AK, Akkoc T, Akdis M, Akdis CA. Role of Treg in immune regulation of allergic diseases. Eur J Immunol. 2010; 40:1232–1240.
6. Boettler T, Spangenberg HC, Neumann-Haefelin C, Panther E, Urbani S, Ferrari C, et al. T cells with a CD4+CD25+ regulatory phenotype suppress in vitro proliferation of virus-specific CD8+ T cells during chronic hepatitis C virus infection. J Virol. 2005; 79:7860–7867.
7. Hazenberg MD, Otto SA, van Benthem BH, Roos MT, Coutinho RA, Lange JM, et al. Persistent immune activation in HIV-1 infection is associated with progression to AIDS. AIDS. 2003; 17:1881–1888.
8. Hunt PW, Landay AL, Sinclair E, Martinson JA, Hatano H, Emu B, et al. A low T regulatory cell response may contribute to both viral control and generalized immune activation in HIV controllers. PLoS One. 2011; 6:e15924
9. Eggena MP, Barugahare B, Jones N, Okello M, Mutalya S, Kityo C, et al. Depletion of regulatory T cells in HIV infection is associated with immune activation. J Immunol. 2005; 174:4407–4414.
10. Oswald-Richter K, Grill SM, Shariat N, Leelawong M, Sundrud MS, Haas DW, et al. HIV infection of naturally occurring and genetically reprogrammed human regulatory T-cells. PLoS Biol. 2004; 2:E198
11. Prendergast A, Prado JG, Kang YH, Chen F, Riddell LA, Luzzi G, et al. HIV-1 infection is characterized by profound depletion of CD161+ Th17 cells and gradual decline in regulatory T cells. AIDS. 2010; 24:491–502.
12. Bi X, Suzuki Y, Gatanaga H, Oka S. High frequency and proliferation of CD4+ FOXP3+ Treg in HIV-1-infected patients with low CD4 counts. Eur J Immunol. 2009; 39:301–309.
13. Nilsson J, Boasso A, Velilla PA, Zhang R, Vaccari M, Franchini G, et al. HIV-1-driven regulatory T-cell accumulation in lymphoid tissues is associated with disease progression in HIV/AIDS. Blood. 2006; 108:3808–3817.
14. Suchard MS, Mayne E, Green VA, Shalekoff S, Donninger SL, Stevens WS, et al. FOXP3 expression is upregulated in CD4T cells in progressive HIV-1 infection and is a marker of disease severity. PLoS One. 2010; 5:e11762
15. Moreno-Fernandez ME, Zapata W, Blackard JT, Franchini G, Chougnet CA. Human regulatory T cells are targets for human immunodeficiency virus (HIV) infection, and their susceptibility differs depending on the HIV type 1 strain. J Virol. 2009; 83:12925–12933.
16. Williams LM, Rudensky AY. Maintenance of the Foxp3-dependent developmental program in mature regulatory T cells requires continued expression of Foxp3. Nat Immunol. 2007; 8:277–284.
17. Chen W, Jin W, Hardegen N, Lei KJ, Li L, Marinos N, et al. Conversion of peripheral CD4+CD25- naive T cells to CD4+CD25+ regulatory T cells by TGF-beta induction of transcription factor Foxp3. J Exp Med. 2003; 198:1875–1886.
18. Fontenot JD, Gavin MA, Rudensky AY. Foxp3 programs the development and function of CD4+CD25+ regulatory T cells. Nat Immunol. 2003; 4:330–336.
19. Allan SE, Crome SQ, Crellin NK, Passerini L, Steiner TS, Bacchetta R, et al. Activation-induced FOXP3 in human T effector cells does not suppress proliferation or cytokine production. Int Immunol. 2007; 19:345–354.
20. Tran DQ, Ramsey H, Shevach EM. Induction of FOXP3 expression in naive human CD4+FOXP3 T cells by T-cell receptor stimulation is transforming growth factor-beta dependent but does not confer a regulatory phenotype. Blood. 2007; 110:2983–2990.
21. Huehn J, Polansky JK, Hamann A. Epigenetic control of FOXP3 expression: the key to a stable regulatory T-cell lineage?. Nat Rev Immunol. 2009; 9:83–89.
22. Janson PC, Winerdal ME, Marits P, Thorn M, Ohlsson R, Winqvist O. FOXP3 promoter demethylation reveals the committed Treg population in humans. PLoS One. 2008; 3:e1612
23. Waterhouse PM, Wang MB, Lough T. Gene silencing as an adaptive defence against viruses. Nature. 2001; 411:834–842.
24. Kauder SE, Bosque A, Lindqvist A, Planelles V, Verdin E. Epigenetic regulation of HIV-1 latency by cytosine methylation. PLoS Pathog. 2009; 5:e1000495
25. Pion M, Jordan A, Biancotto A, Dequiedt F, Gondois-Rey F, Rondeau S, et al. Transcriptional suppression of in vitro-integrated human immunodeficiency virus type 1 does not correlate with proviral DNA methylation. J Virol. 2003; 77:4025–4032.
26. Fang JY, Mikovits JA, Bagni R, Petrow-Sadowski CL, Ruscetti FW. Infection of lymphoid cells by integration-defective human immunodeficiency virus type 1 increases de novo methylation. J Virol. 2001; 75:9753–9761.
27. Mikovits JA, Young HA, Vertino P, Issa JP, Pitha PM, Turcoski-Corrales S, et al. Infection with human immunodeficiency virus type 1 upregulates DNA methyltransferase, resulting in de novo methylation of the gamma interferon (IFN-gamma) promoter and subsequent downregulation of IFN-gamma production. Mol Cell Biol. 1998; 18:5166–5177.
28. Garcia-Merino I, de Las Cuevas N, Jimenez JL, Gallego J, Gomez C, Prieto C, et al. The Spanish HIV BioBank: a model of cooperative HIV research. Retrovirology. 2009; 6:27
29. Lal G, Zhang N, van der Touw W, Ding Y, Ju W, Bottinger EP, et al. Epigenetic regulation of Foxp3 expression in regulatory T cells by DNA methylation. J Immunol. 2009; 182:259–273.
30. Josefowicz SZ, Rudensky A. Control of regulatory T cell lineage commitment and maintenance. Immunity. 2009; 30:616–625.
31. Miyara M, Yoshioka Y, Kitoh A, Shima T, Wing K, Niwa A, et al. Functional delineation and differentiation dynamics of human CD4+ T cells expressing the FoxP3 transcription factor. Immunity. 2009; 30:899–911.
32. Garcia JV, Miller AD. Serine phosphorylation-independent downregulation of cell-surface CD4 by nef. Nature. 1991; 350:508–511.
33. Hoffmann P, Boeld TJ, Eder R, Huehn J, Floess S, Wieczorek G, et al. Loss of FOXP3 expression in natural human CD4+CD25+ regulatory T cells upon repetitive in vitro stimulation. Eur J Immunol. 2009; 39:1088–1097.
34. Kinter A, McNally J, Riggin L, Jackson R, Roby G, Fauci AS. Suppression of HIV-specific T cell activity by lymph node CD25+ regulatory T cells from HIV-infected individuals. Proc Natl Acad Sci U S A. 2007; 104:3390–3395.
35. Foxall RB, Albuquerque AS, Soares RS, Baptista AP, Cavaleiro R, Tendeiro R, et al. Memory and naive-like regulatory CD4+ T cells expand during HIV-2 infection in direct association with CD4+ T-cell depletion irrespectively of viremia. AIDS. 2011; 25:1961–1970.
36. Card CM, Keynan Y, Lajoie J, Bell CP, Dawood M, Becker M, et al. HIV controllers are distinguished by chemokine expression profile and HIV-specific T-cell proliferative potential. J Acquir Immune Defic Syndr. 2012; 59:427–437.
37. Moreno-Fernandez ME, Presicce P, Chougnet CA. Homeostasis and function of regulatory T cells in HIV/SIV infection. J Virol. 2012; 86:10262–10269.
38. Presicce P, Orsborn K, King E, Pratt J, Fichtenbaum CJ, Chougnet CA. Frequency of circulating regulatory T cells increases during chronic HIV infection and is largely controlled by highly active antiretroviral therapy. PLoS One. 2011; 6:e28118
39. Del Pozo-Balado Mdel M, Leal M, Mendez-Lagares G, Pacheco YM. CD4(+)CD25(+/hi)CD127(lo) phenotype does not accurately identify regulatory T cells in all populations of HIV-infected persons. J Infect Dis. 2010; 201:331–335.
40. Youngblood B, Reich NO. The early expressed HIV-1 genes regulate DNMT1 expression. Epigenetics. 2008; 3:149–156.
41. Ji J, Cloyd MW. HIV-1 binding to CD4 on CD4+CD25+ regulatory T cells enhances their suppressive function and induces them to home to, and accumulate in, peripheral and mucosal lymphoid tissues: an additional mechanism of immunosuppression. Int Immunol. 2009; 21:283–294.
42. Wan YY, Flavell RA. Regulatory T-cell functions are subverted and converted owing to attenuated Foxp3 expression. Nature. 2007; 445:766–770.
43. Ouyang W, Beckett O, Ma Q, Li MO. Transforming growth factor-beta signaling curbs thymic negative selection promoting regulatory T cell development. Immunity. 2010; 32:642–653.
44. Sugimoto N, Oida T, Hirota K, Nakamura K, Nomura T, Uchiyama T, et al. Foxp3-dependent and -independent molecules specific for CD25+CD4+ natural regulatory T cells revealed by DNA microarray analysis. Int Immunol. 2006; 18:1197–1209.
45. Veldman C, Pahl A, Beissert S, Hansen W, Buer J, Dieckmann D, et al. Inhibition of the transcription factor Foxp3 converts desmoglein 3-specific type 1 regulatory T cells into Th2-like cells. J Immunol. 2006; 176:3215–3222.
46. Buonaguro L, Tornesello ML, Gallo RC, Marincola FM, Lewis GK, Buonaguro FM. Th2 polarization in peripheral blood mononuclear cells from human immunodeficiency virus (HIV)-infected subjects, as activated by HIV virus-like particles. J Virol. 2009; 83:304–313.
47. Arruvito L, Sabatte J, Pandolfi J, Baz P, Billordo LA, Lasala MB, et al. Analysis of suppressor and nonsuppressor FOXP3+ T cells in HIV-1-infected patients. PLoS One. 2012; 7:e52580
DNA methyltransferase; Foxp3; HIV infection; immune hyperactivation; methylation; suppressive function; Treg
Supplemental Digital Content
© 2013 Wolters Kluwer Health | Lippincott Williams & Wilkins
Highlight selected keywords in the article text.