Clinical Infectious Diseases MAJOR ARTICLE The Genome-wide Methylation Profile of CD4+ T Cells From Individuals With Human Immunodeficiency Virus (HIV) Identifies Distinct Patterns Associated With Disease Progression Sara Moron-Lopez,1 Victor Urrea,1 Judith Dalmau,1 Miguel Lopez,2,3 Maria C. Puertas,1 Dan Ouchi,1 Antonio Gómez,2 Caroline Passaes,4 Beatriz Mothe,1,5,6 Christian Brander,1,6,7 Asier Saez-Cirion,4 Bonaventura Clotet,1,5,6 Manel Esteller,7,8,9,10 Maria Berdasco,2,3 and Javier Martinez-Picado1,6,7 1 AIDS Research Institute IrsiCaixa, Badalona, Spain, 2Cancer Epigenetics Group, Cancer Epigenetics and Biology Program, Bellvitge Biomedical Research Institute, Barcelona, Spain, Downloaded from https://academic.oup.com/cid/article/72/9/e256/5876587 by guest on 15 June 2021 3 Epigenetic Therapies Group, Experimental and Clinical Hematology Program, Josep Carreras Leukaemia Research Institute, Badalona, Spain, 4Institut Pasteur, Unité HIV, Inflammation et Persistence, Paris, France, 5Fundació Lluita Contra la Sida, University Hospital “Germans Trias i Pujol,” Badalona, Spain, 6University of Vic-Central University of Catalonia (UVic-UCC), Barcelona, Spain, 7Catalan Institution for Research and Advanced Studies (ICREA), Barcelona, Spain, 8Josep Carreras Leukaemia Research Institute (IJC), Badalona, Spain, 9Centro de Investigación Biomédica en Red Cancer (CIBERONC), Madrid, Spain, and 10Physiological Sciences Department, School of Medicine and Health Sciences, University of Barcelona (UB), Badalona, Spain Background. Human genetic variation—mostly in the human leukocyte antigen (HLA) and C–C chemokine receptor type 5 (CCR5) regions—explains 25% of the variability in progression of human immunodeficiency virus (HIV) infection. However, it is also known that viral infections can modify cellular DNA methylation patterns. Therefore, changes in the methylation of cytosine- guanine (CpG) islands might modulate progression of HIV infection. Methods. In total, 85 samples were analyzed: 21 elite controllers (EC), 21 subjects with HIV before combination antiretroviral therapy (cART) (viremic, 93 325 human immunodeficiency virus type 1 [HIV-1] RNA copies/mL) and under suppressive cART (cART, median of 17 months, <50 HIV-1 RNA copies/mL), and 22 HIV-negative donors (HIVneg). We analyzed the methylation pattern of 485 577 CpG in DNA from peripheral CD4+ T lymphocytes. We selected the most differentially methylated gene (TNF) and analyzed its specific methyla- tion, messenger RNA (mRNA) expression, and plasma protein levels in 5 individuals before and after initiation of cART. Results. We observed 129 methylated CpG sites (associated with 43 gene promoters) for which statistically significant differ- ences were recorded in viremic versus HIVneg, 162 CpG sites (55 gene promoters) in viremic versus cART, 441 CpG sites (163 gene promoters) in viremic versus EC, but none in EC versus HIVneg. The TNF promoter region was hypermethylated in viremic versus HIVneg, cART, and EC. Moreover, we observed greater plasma levels of TNF in viremic individuals than in EC, cART, and HIVneg. Conclusions. Our study shows that genome methylation patterns vary depending on HIV infection status and progression pro- file and that these variations might have an impact on controlling HIV infection in the absence of cART. Keywords. genome-wide methylation; HIV-1 infection; HIV-1 disease progression; TNF. Human genetic variation—mostly in the human leukocyte changes in the methylation of cytosine-guanine (CpG) islands antigen (HLA) and chemokine receptor 5 (CCR5) regions— could modulate progression of HIV infection through varia- explains 25% of the variability in progression of human immu- tions in transcription and expression of relevant genes. During nodeficiency virus (HIV) infection [1]. Epigenetic chromatin HIV infection, the viral genome integrates into the host cellular modification regulates cellular gene transcription and expres- genome, altering the chromatin structure and therefore poten- sion without changing the DNA sequence [2]. Because viral tially modifying host gene expression. This virus-host interac- infections can modify cellular DNA methylation patterns [3], tion produces changes in the epigenetic environment that affect both the virus and the host cell [3, 4]. Regarding DNA meth- Received 27 March 2020; editorial decision 24 June 2020; accepted 21 July 2020; published ylation, on the one hand, hypermethylation of the HIV 5′ long online July 26, 2020. terminal repeat (LTR) may inhibit initiation of viral transcrip- Correspondence: J. Martínez-Picado, IrisCaixa Foundation, Badalona, Barcelona, Spain (jmpicado@irsicaixa.es). tion and thus favor HIV latency [5–8]. Furthermore, recent data Clinical Infectious Diseases®  2021;72(9):e256–64 suggest that methylation of 5′ LTR of HIV is associated with © The Author(s) 2020. Published by Oxford University Press for the Infectious Diseases control of viral replication in a subset of individuals with HIV Society of America. This is an Open Access article distributed under the terms of the Creative Commons Attribution-NonCommercial-NoDerivs licence (http://creativecommons.org/licenses/ characterized as long-term nonprogressors and/or elite control- by-nc-nd/4.0/), which permits non-commercial reproduction and distribution of the work, in any lers (EC) [9]. On the other hand, cellular DNA methylation may medium, provided the original work is not altered or transformed in any way, and that the work is properly cited. For commercial re-use, please contact journals.permissions@oup.com regulate T-lymphocyte gene expression in order to favor the es- DOI: 10.1093/cid/ciaa1047 tablishment of infection, thus promoting viral replication and e256 • cid 2021:72 (1 May) • Moron-Lopez et al facilitating viral persistence [8]. Specifically, HIV can induce de detailed in Table 1. The specific methylation analysis was based novo methylation of host cell genes through the induction of on a validation cohort of 5 participants with HIV with avail- DNA methyltransferase 1 (DNMT1) in vitro and in vivo [4, 10]. able samples before cART and at 2 time points after initiation In addition, several studies showed differences in genome-wide of suppressive cART (1 and 2 years after initiation). The clin- DNA methylation between individuals with HIV and individ- ical characteristics of the validation cohort at sampling are de- uals who do not have HIV [11–13]. However, there are no data tailed in Supplementary Table 1. For this retrospective study, comparing genome-wide methylation patterns in CD4+ T cells cryopreserved peripheral blood mononuclear cell (PBMC) from HIV-negative individuals with longitudinal samples from samples from HIV-infected and HIV-negative individuals for individuals with HIV with different phenotypes of spontaneous the discovery and the validation cohorts were selected from the viral control and under suppressive combination antiretroviral IrsiCaixa collection. All samples met legal requirements, in- therapy (cART). cluding written informed consent from participants and donors In this context, the present study aimed to evaluate the in for the use of biological samples, and complied with the stipula- vivo genome-wide DNA methylation profile in individuals tions of the Institutional Review Board (PI-13–024). with HIV with different phenotypes of spontaneous viral con- Downloaded from https://academic.oup.com/cid/article/72/9/e256/5876587 by guest on 15 June 2021 trol and under suppressive cART. This approach enabled us to Procedures determine (i) which changes are preferentially associated with CD4+ T cells purified from 85 cryopreserved PBMC samples viremia and/or CD4 depletion (only observed in viremic indi- were analyzed for genome-wide methylation quantification on viduals), (ii) which changes are reversible or nonreversible after an Infinium HumanMethylation450 BeadChip following the viral suppression with cART (comparison between viremic and Illumina Infinium HD Methylation Assay Protocol. Bisulfite cART samples), and (iii) which changes might be associated genomic sequencing of multiple clones, methylation-specific with viral control in the absence of cART (comparison between polymerase chain reaction (PCR), messenger RNA (mRNA) ex- ECs and viremic/cART-treated individuals). pression, and plasma levels of the selected tumor necrosis factor (TNF) gene were performed in samples of the validation cohort. The candidate CpGs contained in the methylation array were MATERIAL AND METHODS included in the PCR amplicon (Supplementary Table 2). The Study Design primer sequences are listed in Supplementary Table 3. More de- In total, 42 individuals living with HIV with controlled infec- tailed procedures are described in Supplementary Materials. tion (EC group, n = 21, defined as undetectable plasma viral load [pVL] in the absence of cART) or uncontrolled infection Statistical Analyses (viremic group, n = 21, defined as detectable pVL > 4 log10 cop/ Methylation raw data normalization was performed using mL before initiation of cART). Paired with the viremic group, minfi, lumi, and associated R packages following Quantile longitudinal samples were also taken after at least 6 months normalization. In the differential methylation analysis be- under effective suppression with cART (cART group). Samples tween cohorts of each CpG, a t test was used to compare from 22 HIV-negative donors (HIVneg group) were used based β-value means between subject groups through pairwise on the cis-gender and age of the groups with HIV. The clin- comparisons, correcting by multiple testing (false discovery ical characteristics of the discovery cohort at sampling are rate). The CpGs selected were those with adjusted values of Table 1. Characteristics of the Discovery Cohort at the Sampling Time Point, by Group HIV Positive Viremic (n = 21) cART (n = 21) EC (n = 21) HIV Negative (n = 22) a Age (years) 32 (28–35) 34 (29–37) 44 (40–50) 31 (29–42) Sex (n, %) Men 21 (100) 21 (100) 8 (38) 4 (18) Women 0 0 12 (57) 9 (41) Missing 0 0 1 (5) 9 (41) CD4+ T-cell counts (cells/μL)a 444 (350–565) 734 (578–808) 645 (505–1001) ND CD4/CD8 ratioa 0·5 (0·3–0·6) 1 (0·7–1·1) ND ND Plasma viremia (HIV RNA copies/mL) 9·4 × 104 (0·4–1·4 × 105) UD UD NA Time since diagnosis (years)a 1 (0·8–1·3) 2·8 (2·0–3·1) 17 (8–20) NA Time since initiation of cART (years)a 0 1·4 (1·2–1·5) 0 NA Abbreviations: cART, combination antiretroviral therapy; EC, elite controller; HIV, human immunodeficiency virus; NA, not applicable; ND, no data; UD, undetectable (either with 25, 40, or 50 HIV-1 RNA copies/mL threshold). a Median (interquartile range [IQR]). Host DNA Methylation and HIV Progression • cid 2021:72 (1 May) • e257 P < .05 and an absolute methylation differential value of >0.05 hypomethylation being observed only under suppressive (>5%). Hypergeometric test was used to determine differen- cART, thus suggesting that BCL9 methylation might be af- tially methylated gene promoter regions by comparing the fected by administration of cART. Furthermore, we ob- number of differentially methylated CpGs and the number served hypomethylation of tripartite motif containing of CpGs tested in each region. KEGG pathway enrichment 69 (TRIM69) and integrin subunit beta 2 (ITGB2) (all analysis was performed taking into account only genes with a padj < 1 × 10−7) in EC when compared with viremic individ- differentially methylated promoter region. Wilcoxon signed uals, suggesting that the expression of these genes might be rank test (paired comparisons: viremic vs cART) or Mann- upregulated (hypomethylated) in EC and, therefore, have Whitney test (nonpaired comparisons: viremic vs EC and an impact on control of HIV infection. We also observed HIVneg, and EC vs HIVneg) were used to analyzed tumor hypermethylation of lymphotoxin beta (LTB) (padj < 1 × 10−7) necrosis factor (TNF) specific methylation, mRNA expres- and hypomethylation of transducing-like enhancer family sion, and plasma levels using GraphPad Prism (v5.0). member 1 (TLE1) (padj = 1·19 × 10−7) when we compared vi- remic vs HIVneg and EC, thus indicating a viremia-driven RESULTS methylation pattern that was not reverted under suppressive Downloaded from https://academic.oup.com/cid/article/72/9/e256/5876587 by guest on 15 June 2021 cART. Moreover, we observed that cluster of differentiation Genome-wide Differences in Methylation Between Viremic, cART- Suppressed, and EC Individuals With HIV and HIVneg Individuals 2 (CD2) was hypomethylated both in EC (padj = 3·25 × 10−4) In order to determine whether HIV infection affects host cel- and in cART (padj = 2·98 × 10−5) compared with viremic in- lular genome-wide methylation, and the steadiness of such dividuals, suggesting upregulation of CD2 in virally sup- potential modifications, we compared the methylation level pressed CD4+ T cells and therefore an association between of 485 577 CpG sites in CD4+ T cells from viremic and paired CD2 expression with HIV latent infection. cART-suppressed, EC, and HIV-negative individuals. Regarding Regarding the association between these 7 genes of interest methylated CpG sites for which statistically significant differ- and HIV, we analyzed the STRING protein interaction and ences were recorded, we observed 129 differentially methylated previous bibliography related to HIV (Figure 2C). Interaction CpGs between viremic and HIVneg individuals (median [min, with HIV was previously reported for TNF, LTB, ITGB2, CD2, max] methylation difference: 7.3 [−15.6, 16.6]%), 162 between TLE1, and BCL9. Furthermore, we observed that TNF, LTB, viremic and cART-suppressed individuals (6.5 [5.0, 12.8]%), ITGB2, and CD2 interact with each other and are associated and 441 between viremic and EC individuals (6.3 [−16.4, with the immune response, whereas TLE1 and BCL9 interact 13.8]%). However, we did not observe statistically significant with each other and are associated with gene expression, thus differences in methylated CpG sites between EC and HIVneg indicating that HIV infection might affect the methylation pat- individuals (Figure 1A–D). tern of immune response-related genes and transcription fac- To characterize the genes associated with these differen- tors. Interestingly, no interaction has been reported between tially methylated CpG sites, we analyzed the CpG located in HIV and TRIM69, which is involved in induction of apoptosis gene regulatory regions (5′ UTR, TSS, and the first exon were and, in this study, was hypomethylated in EC, thus suggesting included) and with a methylation difference >5% (Figure 2A). that its expression might be associated with control of HIV in Specifically, we detected statistically significant differences in the absence of cART. methylation between viremic and HIVneg individuals in 43 Overall, these data suggest that HIV infection modifies genes (Supplementary Table 4), between viremic and cART- levels of cellular host genome methylation and that meth- suppressed individuals in 55 genes (Supplementary Table ylation levels might be associated with progression of HIV 5), and between viremic and EC individuals in 163 genes infection. (Supplementary Table 6). Within the top 15 genes per comparison, we observed The T-Cell Receptor Signaling Pathway Is Enriched in Differentially that TNF was the most differentially methylated gene Methylated CpG Sites in Viremic Individuals when comparing viremic versus HIVneg and viremic To determine the cellular pathways associated with statistically versus EC (padj < 1 × 10−25), with hypermethylation ob- significant differences in methylation at CpG sites located in a served in the viremic individuals (Figure 2B). We also promotor region and with a fold-change >5%, we performed a observed hypermethylation in TNF promoter when com- KEGG pathway enrichment analysis using the previously listed paring longitudinal samples from individuals with HIV be- genes related to these CpG sites. We observed 58 differentially fore and after viral suppression with cART (padj < 0·001), enriched cellular pathways (Supplementary Table 7), from thus indicating that viremia-driven TNF hypermethylation which 5 were enriched in the comparisons of viremic versus was reverted. However, when we compared viremic versus cART-suppressed, versus EC individuals, and versus HIVneg; cART (padj = 1·68 × 10−8), the greatest difference in meth- the “T-cell receptor signaling pathway” was the most signifi- ylation was in BCL9 transcription coactivator (BCL9) with cantly enriched KEGG pathway (Supplementary Table 7). e258 • cid 2021:72 (1 May) • Moron-Lopez et al A B TLE1 HIVneg Viremic vs. vs. Viremic cART CD2 BCL9 Downloaded from https://academic.oup.com/cid/article/72/9/e256/5876587 by guest on 15 June 2021 TNF TNF LTB C D TRIM69 ITGB2 TLE1 Viremic HIVneg vs. vs. EC EC CD2 TNF LTB Methylation level CG density 0.0 0.2 0.4 0.6 0.8 1.0 0 36 72 108 144 180 216 252 288 324 360 Hypomethylation Hypermethylation #CG Figure 1. Genome-wide methylation levels by CpG site. (A) HIV-negative (HIVneg) vs viremic, (B) viremic vs cART, (C) viremic vs EC, and (D) HIVneg vs EC. From inside to outside: (i) heatmap of CpGs with significant adjusted P-value and differential methylation over 5%, following the order of the group comparison (no significant differences in methylation of CpG sites were detected in HIVneg vs EC, D); (ii) Manhattan plot of –log10(P-value) for CpG sites with P-value thresholds at 10−5 and 10−7; and (iii) chromo- somes. CpG sites for which differences in methylation were statistically significant are shown in blue (hypomethylated) and red (hypermethylated). The density of CpG sites per chromosome is color-coded. Abbreviations: cART, combination antiretroviral therapy; CpG, cytosine-guanine; EC, elite controllers; HIV, human immunodeficiency virus; LTB, lymphotoxin beta; TNF, tumor necrosis factor. Greater Promoter TNF Methylation as a Counteraction of TNF levels of TNF in viremic individuals than in cART-suppressed, Overexpression in Viremic Individuals EC, and HIV-negative individuals, as well as in EC versus To characterize the effect of HIV viremia on methylation, HIVneg (Figure 3A). These data suggest that hypermethylation mRNA expression, and plasma levels of TNF, we quantified the of TNF promoter might be a homeostatic mechanism for nor- plasma level of TNF in the discovery cohort, and the methyl- malization of TNF production and therefore immune activa- ation, mRNA expression, and plasma levels of TNF in longi- tion due to HIV infection. In the validation cohort, we did tudinal samples (before and after initiation of cART) from 5 not observe significant changes in specific DNA methylation independent individuals with HIV. We observed greater plasma in the TNF promoter region or mRNA expression in TNF in Host DNA Methylation and HIV Progression • cid 2021:72 (1 May) • e259 A CD4+ T cells from CD4+ T cells from PLWH HIV negative donors Viremic - cART (n=21) (n=22) EC (n=21) vs Genome-wide methylation patterns (485,577 CpG) Methylation analysis (FDR<0.05) Downloaded from https://academic.oup.com/cid/article/72/9/e256/5876587 by guest on 15 June 2021 Differentially methylated CpG Viremic vs EC vs HIVneg cART EC HIVneg # CpG 129 162 441 0 Gene enrichment analysis (methylation difference >5%; CpG in gene promoter) Differentially methylated genes Viremic vs EC vs HIVneg cART EC HIVneg # Genes 43 55 163 0 Top 15 genes per comparison B Methylation level of genes of interest C Interaction between genes of interest and HIV Viremic vs EC vs HIVneg cART EC HIVneg CD2 TNF ITGB2 BCL9 TRIM69 TRIM69 ITGB2 LTB TNF TLE1 CD2 BCL9 TLE1 Hypo- Hyper- L LTB Methylation level Protein-protein interaction Protein-HIV interaction Methylation level in Vire Viremic vs any other group from curated databases previously reported experimentally determined unknown Hypo- Hyper- textmining co-expression protein homology Figure 2. Analysis pipeline overview, methylation level of genes of interest, and interaction between genes of interest and HIV. A, Schematic representation of the bi- oinformatics analysis pipeline overview, (B) the methylation level of the differentially methylated genes of interest, and (C) the interaction between the genes of interest (using STRING) and HIV (based on previous bibliography). The methylation level is shown in blue (hypomethylated) and red (hypermethylated). Abbreviations: cART, combined antiretroviral therapy; EC, elite controllers; FDR, false discovery rate; HIV, human immunodeficiency virus; LTB, lymphotoxin beta; PLWH, people living with HIV; TNF, tumor necrosis factor. e260 • cid 2021:72 (1 May) • Moron-Lopez et al A [TNF] in plasma * ** 15 *** *** [TNF] (pg/ml plasma) 10 5 0 =2 ic =1 eg (n EC =2 T 9) (n rem (n AR (n Vn 1) 2) 0) =1 c I i H V Downloaded from https://academic.oup.com/cid/article/72/9/e256/5876587 by guest on 15 June 2021 B TNF methylation C TNF expression D [TNF] in plasma Fold change induction (log2) 40 2 10 0.06 [TNF] (pg/ml plasma) 8 30 % Methylation 6 20 1 4 10 2 0 0.5 0 BL BL T1 T2 BL T2 T1 T1 T2 AR AR AR AR AR AR Figure 3. Association between HIV infection, cART, and disease progression on TNF production. A, Plasma TNF level in HIV-negative, viremic, cART, and EC individuals from the discovery cohort (n = 12 HIVneg, n = 20 viremic, n = 21 cART, and n = 19 EC). B, Levels of TNF-specific methylation, (C) mRNA, and (D) plasma in the validation co- hort (n = 5, samples before [BL, baseline], and 1 and 2 years after initiation of cART [ART1 and ART2]). Solid symbols represent undetectable values. The statistical analysis was performed using the Wilcoxon signed rank test (paired comparisons: viremic vs cART) or Mann-Whitney test (nonpaired comparisons: viremic vs EC and HIVneg, and EC vs HIVneg) [GraphPad Prism (v5.0)]. Abbreviations: cART, combined antiretroviral therapy; EC, elite controllers; HIV, human immunodeficiency virus; mRNA, messenger RNA; TNF, tumor necrosis factor. longitudinal samples pre- and post-viral suppression by cART Several studies performed in vitro and in vivo have analyzed (Figure 3B, C). However, we observed a decrease in the plasma how HIV affects host epigenetic mechanisms [11, 13, 15, 16]. level of TNF after initiation of cART (Figure 3D). These results On the one hand, viral infection alters the expression of cellular corroborate that initiation of cART, which suppresses HIV vi- genes that modify chromatin structure. On the other hand, the remia, helps to decrease plasma TNF levels [14] and therefore viral genome itself is modified by some of these mechanisms to can partially contribute to the reduction of global immune ac- regulate its level of transcription [5]. Moreover, lifestyle (eg, nu- tivation under suppressive cART. trition, physical activity, working habits, smoking, and alcohol consumption) and the administration of drugs, such as cART, may influence epigenetic mechanisms [17]. DISCUSSION In our study, we observed 129 differentially methylated CpG The present study is the first to our knowledge to evaluate sites in viremic versus HIVneg individuals, 162 sites in viremic the genome-wide DNA methylation profile in vivo in periph- versus cART individuals, and 441 sites in viremic versus EC eral CD4+ T cells from HIV-negative individuals and indi- individuals. These results confirm not only that HIV infection viduals with HIV with different phenotypes of viral control modifies the DNA methylation profile of the target cells in vivo (EC) and degrees of disease progression (viremic and paired [11–13] but also that these epigenetic modifications differ ac- cART-suppressed and EC individuals). We observed differen- cording to HIV disease progression status and control of infec- tial genome-wide patterns depending on spontaneous ability tion [9]. to control HIV replication and on whether or not suppressive Regarding specific genes, we found that hypermethylation cART had been initiated. of the TNF gene was a differential hallmark of viremic Host DNA Methylation and HIV Progression • cid 2021:72 (1 May) • e261 individuals. Several studies have shown that TNF and TNF re- therefore helping to detect and eliminate HIV-infected cells, ceptor superfamily members have a role in the pathogenesis of which could contribute to the lower reservoir levels described HIV, affecting different stages of the HIV replication cycle and in EC. Interestingly, none of the abovementioned genes has ever being able to increase HIV transcription through activation been related to control of HIV infection or disease progression. of the NF-κB signaling pathway [18]. Moreover, it has been Therefore, further investigation of these genes and their role in shown that expression of TNF is regulated by DNA methyla- HIV infection could prove helpful for deciphering the mech- tion and other mechanisms [19], and that TNF acts as a link anisms that may regulate HIV control in EC, controllers, and between T-cell dysfunction and chronic inflammation during long-term nonprogressors. chronic viral infection [20]. The hypermethylation of the TNF BCL9, which is involved in WNT pathway and required for promoter region and greater plasma levels in viremic individ- HIV transcription [37], was hypermethylated in the viremic uals we observed might constitute a homeostatic mechanism samples compared with cART-suppressed samples, whereas its for downregulation of TNF expression and plasma levels in methylation pattern in viremic individuals was similar to that of order to decrease immune activation and HIV expression. HIVneg donors, suggesting that its expression might be lower in On the other hand, TNF produced by other cell types, such viremic individuals as a mechanism for reducing replication of Downloaded from https://academic.oup.com/cid/article/72/9/e256/5876587 by guest on 15 June 2021 as CD8+ T cells, monocytes, and dendritic cells, which we did HIV or as a direct effect of cART. CD2 is a surface antigen that not include in this study, should be further characterized in interacts with LFA3 (CD58) on antigen-presenting cells to opti- future studies. mize immune recognition, and its expression in resting memory With respect to differences in methylation between viremic CD4+ T cells has been related to latently HIV-infected cells [34]. and EC individuals, we observed that the genes TRIM69, In contrast, CD2 has been positively associated with the expres- and ITGB2, were hypomethylated in EC. These results sug- sion of restriction factors [32], and its hypermethylation in vi- gest that the expression of these genes might be upregulated remic individuals was downregulated by cART, suggesting that and therefore play a role in the control of HIV replication in its increased expression might favor HIV replication. the absence of cART. To our knowledge, TRIM69, which en- The limitations of the study should be acknowledged. First, a codes a member of the RING-B-box-coiled-coil family and low number of individuals was analyzed in both the discovery which, when overexpressed, induces apoptosis, and although and the validation cohorts. This may explain why we did not its DNA methylation has been associated with HIV frailty observe statistically significant differences in the methylation of [21], it has never been related to HIV replication or pathogen- CpG sites when comparing EC with HIVneg. Second, although esis. Its hypomethylation in EC may increase its expression in cART may have an impact on epigenetics, the low number of CD4+ T cells and therefore in HIV-infected cells, causing spe- individuals and the different cART regimens they were taking cific apoptosis of these cells and potentially leading to better prevented us from determining changes in DNA methylation control of HIV infection. Interactions between the HIV repli- related to the specific antiretroviral drugs used. Third, although cation cycle and the LTB, ITGB2, CD2, and TLE1 genes have age and sex bias are undesirable, there is no evidence of a strong already been described [22–35]. ITGB2 encodes an integrin influence of either variable on the methylation level of our can- beta chain, which plays an important role in the immune re- didate genes. Moreover, most aging-related studies focus on sponse by helping leukocytes gather at sites of infection or epigenetic reprogramming during early development [38] or injury, where they are needed to contribute to the immune re- on cohorts with considerable variation in age that exceed the sponse. Hypomethylation of ITGB2 in EC may upregulate their 10 years found among some of our study groups. As for the sex expression, thus enhancing the immune response against HIV composition of our cohorts, we excluded all CpG located in the and potentially helping to control HIV infection in the absence X chromosome and in imprinted genes [39]. In this sense, little of cART. is known about sex-specific variations in methylation patterns The methylation pattern found when we compared viremic for sequences of coding genes in autosomal chromosomes. versus HIVneg and EC, and consisting of LTB hypermethylation Finally, as latently infected cells cannot be differentiated from and TLE1 hypomethylation, defines a hallmark of pathogenic uninfected cells and represent 1 in 1000 total CD4 T cells [40], HIV infection not reverted by cART. LTB is a type II mem- we were not able to specifically analyze the infected peripheral brane protein of the TNF family and an inducer of the inflam- CD4+ T-cell subset. Thus, noninfected cells might dilute the matory response system and is involved in the development changes in host DNA methylation. However, by analyzing total of lymphoid tissue. TLE1 is a transcription factor that inhibits CD4+ T cells, we can assume that the changes observed in the NF-κB and Wnt signaling pathways, which are involved in DNA methylation patterns may be due to HIV infection itself activation of HIV transcription [36]. Conversely, the lack of and to bystander effects in noninfected cells. hypomethylation of TLE1 in EC may prevent increased ex- Data on changes in host genes after in vivo infection are lim- pression, thus decreasing its impact on the NF-κB and Wnt ited, thus highlighting the need for more comprehensive global signaling pathways, enhancing HIV antigen expression, and studies. In this regard, single-cell analysis techniques might e262 • cid 2021:72 (1 May) • Moron-Lopez et al help to isolate the several types of cells representing the im- 5. Kauder SE, Bosque A, Lindqvist A, Planelles V, Verdin E. Epigenetic regulation of HIV-1 latency by cytosine methylation. PLoS Pathog 2009; 5:e1000495. mune system and interacting with the virus and to compare 6. Coull JJ, Romerio F, Sun JM, et al. The human factors YY1 and LSF repress the changes in gene and protein expression, as well as epigenetic human immunodeficiency virus type 1 long terminal repeat via recruitment of histone deacetylase 1. J Virol 2000; 74:6790–9. modifications. 7. Williams SA, Chen LF, Kwon H, Ruiz-Jarabo CM, Verdin E, Greene WC. Our findings confirm that HIV infection modifies host ge- NF-kappaB p50 promotes HIV latency through HDAC recruitment and repres- sion of transcriptional initiation. EMBO J 2006; 25:139–49. nome methylation patterns in vivo and that these patterns differ 8. Blazkova J, Trejbalova K, Gondois-Rey F, et al. CpG methylation controls reactiv- and may play a role in progression of HIV infection. ation of HIV from latency. PLoS Pathog 2009; 5:e1000554. 9. Palacios JA, Pérez-Piñar T, Toro C, et al. Long-term nonprogressor and elite con- Supplementary Data troller patients who control viremia have a higher percentage of methylation in their HIV-1 proviral promoters than aviremic patients receiving highly active an- Supplementary materials are available at Clinical Infectious Diseases online. tiretroviral therapy. J Virol 2012; 86:13081–4. Consisting of data provided by the authors to benefit the reader, the posted 10. Bogoi RN, de Pablo A, Valencia E, et al. Expression profiling of chromatin- materials are not copyedited and are the sole responsibility of the authors, so modifying enzymes and global DNA methylation in CD4+ T cells from patients questions or comments should be addressed to the corresponding author. with chronic HIV infection at different HIV control and progression states. Clin Epigenetics 2018; 10:20. 11. Zhang Y, Li SK, Tsui SK. Genome-wide analysis of DNA methylation associated with Notes HIV infection based on a pair of monozygotic twins. Genom Data 2015; 6:12–5. Downloaded from https://academic.oup.com/cid/article/72/9/e256/5876587 by guest on 15 June 2021 Acknowledgments. The authors acknowledge the commitment of the par- 12. Zhang Y, Li SK, Yi Yang K, et al. Whole genome methylation array reveals the ticipants, which made the study possible. S. M.-L., J. M.-P., J. D., M. E., and down-regulation of IGFBP6 and SATB2 by HIV-1. Sci Rep 2015; 5:10806. M. B. conceived and designed the study. B. M., C. B., and B. C. contributed 13. Zhang X, Justice AC, Hu Y, et al. Epigenome-wide differential DNA methylation to the design of the clinical cohorts. S. M.-L., M. C. P., M. L., and C. P. per- between HIV-infected and uninfected individuals. Epigenetics 2016; 11:750–60. formed experiments. A. S.-C. contributed to the experiments. S. M.-L., V. U., 14. Brazille P, Dereuddre-Bosquet N, Leport C, et al. Decreases in plasma TNF- D. O., and A. G. analyzed the data. S. M.-L., M. B., and J. M.-P. interpreted alpha level and IFN-gamma mRNA level in peripheral blood mononuclear cells (PBMC) and an increase in IL-2 mRNA level in PBMC are associated with ef- the data. S. M.-L. and J. M.-P. wrote the manuscript. All the authors have read fective highly active antiretroviral therapy in HIV-infected patients. Clin Exp and edited the final version of the manuscript and accept its content. Immunol 2003; 131:304–11. Financial support. J. M.-P.’s research group is sponsored in part by the 15. Britton LM, Sova P, Belisle S, et al. A proteomic glimpse into the initial global ep- Spanish Ministry of Science, Innovation and Universities and the European igenetic changes during HIV infection. Proteomics 2014; 14:2226–30. Regional Development Fund through grant SAF2016-80033-R (MINECO/ 16. Maricato JT, Furtado MN, Takenaka MC, et al. Epigenetic modulations in acti- FEDER, UE), the Spanish AIDS network “Red Temática Cooperativa de vated cells early after HIV-1 infection and their possible functional consequences. Investigación en SIDA,” funded by Instituto de Salud Carlos III through the PLoS One 2015; 10:e0119234. projects “RD12/0017/0002 and RD16/0025/0041” (cofunded by European 17. Alegría-Torres JA, Baccarelli A, Bollati V. Epigenetics and lifestyle. Epigenomics Regional Development Fund/European Social Fund) “Investing in your fu- 2011; 3:267–77. 18. Kumar A, Abbas W, Herbein G. TNF and TNF receptor superfamily members ture”), and Grifols. S. M.-L. was supported by an AGAUR-2013FI_B 00275 in HIV infection: new cellular targets for therapy? Mediators Inflamm 2013; PhD fellowship from the Secretariat of Universities and Research of the 2013:484378. Government of Catalonia. C. B. and B. M. were supported by grants from 19. Sullivan KE, Reddy AB, Dietzmann K, et al. Epigenetic regulation of tumor ne- the HIVACAT program and the European Union’s Horizon 2020 research crosis factor alpha. Mol Cell Biol 2007; 27:5147–60. and innovation programme under grant agreement no. 681137 and a grant 20. Beyer M, Abdullah Z, Chemnitz JM, et al. Tumor-necrosis factor impairs CD4+ by the National Institute of Allergy and Infectious Diseases of the National T cell-mediated immunological control in chronic viral infection. Nat Immunol Institutes of Health under award number PO1-AI131568. M. C. P. reports 2016; 17:593–603. grants from Gilead Sciences, outside the submitted work. B. M. reports con- 21. Zhang X, Hu Y, Justice AC, et al. DNA methylation signatures of illicit drug injec- sulting fees from Aelix Therapeutics, SL, and speaking fees from Gilead, tion and hepatitis C are associated with HIV frailty. Nat Commun 2017; 8:2243. 22. Bettaccini AA, Baj A, Accolla RS, Basolo F, Toniolo AQ. Proliferative activity of MSD, and Viiv, outside the submitted work. C. B. reports being CSO and extracellular HIV-1 Tat protein in human epithelial cells: expression profile of share holder of Aelix Therapeutics, outside the submitted work. A. S. pathogenetically relevant genes. BMC Microbiol 2005; 5:20. C. reports research grants from MSD, and speaker fees from MSD, Viiv 23. Tardif MR, Gilbert C, Thibault S, Fortin JF, Tremblay MJ. LFA-1 antagonists as Healthcare, and Janssen, outside the submitted work. B. C. reports grants agents limiting human immunodeficiency virus type 1 infection and transmis- from Merck (MSD), Gilead Sciences, Viiv Healthcare; HIV therapeutic sion and potentiating the effect of the fusion inhibitor T-20. Antimicrob Agents vaccine for Aelix Therapeutics; and Antibodies against HIV for AlbaJuna Chemother 2009; 53:4656–66. Therapeutics, outside the submitted work. M. E. reports personal fees from 24. Valentin A, Lundin K, Patarroyo M, Asjö B. The leukocyte adhesion glycoprotein Ferrer International and Quimatryx, outside the submitted work. J. M. P. re- CD18 participates in HIV-1-induced syncytia formation in monocytoid and T ports funding from AbiVax, AstraZeneca, Gilead, Grifols, Merck, and Viiv cells. J Immunol 1990; 144:934–7. 25. Wilen CB, Tilton JC, Doms RW. HIV: cell binding and entry. Cold Spring Harb Healthcare; and personal fees from AbiVax, Gilead, Janssen, Merck, and Perspect Med 2012; 2:a006866. Viiv Healthcare, outside the submitted work. 26. Shrikant P, Benos DJ, Tang LP, Benveniste EN. HIV glycoprotein 120 enhances Potential conflicts of interest. The authors: No reported conflicts of intercellular adhesion molecule-1 gene expression in glial cells. Involvement of interest. All authors have submitted the ICMJE Form for Disclosure of Janus kinase/signal transducer and activator of transcription and protein kinase Potential Conflicts of Interest. C signaling pathways. J Immunol 1996; 156:1307–14. 27. Van Grol J, Subauste C, Andrade RM, Fujinaga K, Nelson J, Subauste CS. HIV-1 inhibits autophagy in bystander macrophage/monocytic cells through Src-Akt References and STAT3. PLoS One 2010; 5:e11733. 1. McLaren PJ, Coulonges C, Bartha I, et al. Polymorphisms of large effect explain 28. Lafrenie RM, Wahl LM, Epstein JS, Hewlett IK, Yamada KM, Dhawan S. HIV- the majority of the host genetic contribution to variation of HIV-1 virus load. 1-Tat modulates the function of monocytes and alters their interactions with Proc Natl Acad Sci USA 2015; 112:14658–63. microvessel endothelial cells: a mechanism of HIV pathogenesis. J Immunol 2. Lindahl T. DNA methylation and control of gene expression. Nature 1981; 1996; 156:1638–45. 290:363–4. 29. Poggi A, Carosio R, Spaggiari GM, et al. NK cell activation by dendritic cells is 3. Galvan SC, García Carrancá A, Song J, Recillas-Targa F. Epigenetics and animal dependent on LFA-1-mediated induction of calcium-calmodulin kinase II: inhi- virus infections. Front Genet 2015; 6:48. bition by HIV-1 Tat C-terminal domain. J Immunol 2002; 168:95–101. 4. Youngblood B, Reich NO. The early expressed HIV-1 genes regulate DNMT1 ex- 30. Barrero CA, Datta PK, Sen S, et al. HIV-1 Vpr modulates macrophage metabolic pression. Epigenetics 2008; 3:149–56. pathways: a SILAC-based quantitative analysis. PLoS One 2013; 8:e68376. Host DNA Methylation and HIV Progression • cid 2021:72 (1 May) • e263 31. Pelchen-Matthews A, Giese S, Mlčochová P, Turner J, Marsh M. β2 integrin adhe- 36. Karn J, Stoltzfus CM. Transcriptional and posttranscriptional regulation of HIV-1 sion complexes maintain the integrity of HIV-1 assembly compartments in pri- gene expression. Cold Spring Harb Perspect Med 2012; 2:a006916. mary macrophages. Traffic 2012; 13:273–91. 37. Brass AL, Dykxhoorn DM, Benita Y, et al. Identification of host proteins re- 32. Bolduan S, Koppensteiner H, Businger R, et al. T cells with low CD2 levels express quired for HIV infection through a functional genomic screen. Science 2008; reduced restriction factors and are preferentially infected in therapy naive chronic 319:921–6. HIV-1 patients. J Int AIDS Soc 2017; 20:21865. 38. Martino DJ, Tulic MK, Gordon L, et al. Evidence for age-related and individual- 33. Asmuth DM, Utay NS, Pollard RB. Peginterferon α-2a for the treatment of HIV specific changes in DNA methylation profile of mononuclear cells during early infection. Expert Opin Investig Drugs 2016; 25:249–57. immune development in humans. Epigenetics 2011; 6:1085–94. 34. Iglesias-Ussel M, Vandergeeten C, Marchionni L, Chomont N, Romerio F. High 39. El-Maarri O, Becker T, Junen J, et al. Gender specific differences in levels of DNA levels of CD2 expression identify HIV-1 latently infected resting memory CD4+ methylation at selected loci from human total blood: a tendency toward higher T cells in virally suppressed subjects. J Virol 2013; 87:9148–58. methylation levels in males. Hum Genet 2007; 122:505–14. 35. Kim DY, Kwon E, Hartley PD, et al. CBFβ stabilizes HIV Vif to counteract 40. Ho YC, Shan L, Hosmane NN, et al. Replication-competent noninduced pro- APOBEC3 at the expense of RUNX1 target gene expression. Mol Cell 2013; viruses in the latent reservoir increase barrier to HIV-1 cure. Cell 2013; 49:632–44. 155:540–51. Downloaded from https://academic.oup.com/cid/article/72/9/e256/5876587 by guest on 15 June 2021 e264 • cid 2021:72 (1 May) • Moron-Lopez et al