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Methylome-wide Analysis of Chronic HIV Infection Reveals Five-Year Increase in Biological Age and Epigenetic Targeting of HLA..... "our study provides a robust estimate of a 5-year age advancement in HIV/cART individuals....increasing expected mortality risk by 19%"
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Highlights
• •Methylome-wide analysis of HIV chronically infected, cART treated individuals
• •HIV+ individuals have an epigenetic age 4.9 years older than healthy controls
• •HLA locus is hypomethylated in HIV+ individuals
• •HIV methylation aging signature is validated in purified cells
Summary
HIV-infected individuals are living longer on antiretroviral therapy, but many patients display signs that in some ways resemble premature aging. To investigate and quantify the impact of chronic HIV infection on aging, we report a global analysis of the whole-blood DNA methylomes of 137 HIV+ individuals under sustained therapy along with 44 matched HIV- individuals. First, we develop and validate epigenetic models of aging that are independent of blood cell composition. Using these models, we find that both chronic and recent HIV infection lead to an average aging advancement of 4.9 years, increasing expected mortality risk by 19%. In addition, sustained infection results in global deregulation of the methylome across >80,000 CpGs and specific hypomethylation of the region encoding the human leukocyte antigen locus (HLA). We find that decreased HLA methylation is predictive of lower CD4 / CD8 T cell ratio, linking molecular aging, epigenetic regulation, and disease progression.
"our study provides a robust estimate of a 5-year age advancement in HIV/cART individuals....increasing expected mortality risk by 19%.....chronic HIV infection is accompanied by a tangible gerontological phenotype......may influence regulation of HLA gene expression and also encompasses signatures of aging.....These findings support a link between HIV infection and aging (Rickabaugh et al., 2015), as quantitatively measured by epigenomic profiling (Figure 1D).......patients more recently infected with HIV (<5 years) had no significant difference in age advancement from those patients with chronic (>12 years) infection----These findings lend support to the theory that age advancement occurs early in the course of disease as a consequence of acute infection or reaction to drug treatment.....we found that the age advancement of HIV+ individuals was negatively correlated with the ratio of CD4+ / CD8+ T lymphocytes......we had observed multiple effects of HIV on the methylome including changes in cellular composition, age advancement, and a general increase in methylome disorder. These differences might be particularly pronounced in HIV-infected patients, some of whom have low CD4+ T cell counts......This study is based on the same epigenetic model of biological aging as many others, including recent reports associating epigenetic aging with Down's Syndrome (Horvath et al., 2015), traumatic stress (Boks et al., 2015), and even all-cause mortality (Marioni et al., 2015)."
[ What is DNA Methylation? https://en.wikipedia.org/wiki/DNA_methylation......DNA methylation is essential for normal development and is associated with a number of key processes, and is associated with a number of key processes including genomic imprinting, X-chromosome inactivation, repression of repetitive elements, aging and carcinogenesis......There is a global loss of DNA methylation during aging....DNA methylation can stably alter the expression of genes in cells as cells divide and differentiate from embryonic stem cells into specific tissues. The resulting change is normally permanent and unidirectional, preventing a cell from reverting to a stem cell or converting into a different cell type.....Methylation modifications that regulate gene expression are usually heritable......Aberrant DNA methylation patterns - hypermethylation and hypomethylation compared to normal tissue - have been associated with a large number of human malignancies......Epigenetic modifications such as DNA methylation have been implicated in cardiovascular disease......Two of the cell types targeted for DNA methylation polymorphisms are monocytes and lymphocytes, which experience an overall hypomethylation" ]
"Thus far, we had observed multiple effects of HIV on the methylome, including changes in cellular composition, age advancement, and a general increase in methylome disorder. We next sought to determine whether there are specific genomic regions for which the methylation state is particularly associated with HIV infection, independent of aging or other factors (Experimental Procedures). Toward this aim, we conducted an analysis of HIV-associated CpG markers independent of disorder or age, controlling for the effects of cellular composition. Analysis of the whole-blood data identified a single genomic region that was enriched in CpG markers associated with HIV; this region, consisting of 10 Mb on chromosome 6 including histone gene cluster 1 and the entire HLA locus, had particularly reduced methylation levels in HIV+ cases as compared to HIV- controls (p < 10-10, Figure 5A; Experimental Procedures). HLA genes encode the Major Histocompatibility Complexes (MHCs), the key antigen-presenting molecules that govern the acquired immune response and impact innate immunity (Figure 5B) (Goulder and Walker, 2012). We found that the differentially methylated markers surround the rs2395029 variant, for which common genetic variation has been repeatedly implicated in HIV host control (Figures 5C and 5D) (Fellay et al., 2007, International HIV Controllers Study et al., 2010). Examination of this locus in the validation samples of purified neutrophils and CD4+ T cells identified the HCP5 gene body as particularly differentially methylated in neutrophils (Figures 5E, 5F, and S6). As further evidence that the observed changes are functional, we found that the amount of methylation at this gene was correlated with a patient's CD4+/CD8+ T cell ratio (Figure S6). Taken together, these results indicate that the HLA locus is likely differentially methylated across blood cell types and also changes within individual cell types in response to HIV. An intriguing interpretation of our results is that some of the previously reported changes in HLA expression and corresponding HIV control (Apps et al., 2013) are attributable to methylation dynamics."
......."we found a striking association with methylation in the HIV+ patients relative to healthy controls (p < 10-100, Figure 1B). Further analysis of these sites found a positive association of the first principal component with both age and HIV status (Figure 1C; Table S3, association by multivariate linear model p < 10-8). These findings support a link between HIV infection and aging (Rickabaugh et al., 2015), as quantitatively measured by epigenomic profiling (Figure 1D)......We have previously reported that age-associated markers in older subjects tend away from a fully methylated or unmethylated state and instead move toward disorder (with a methylation fraction of 50% representing complete disorder) (Hannum et al., 2012). We found that HIV-infected patients displayed a similar trait: among markers associated with HIV, 66% tended toward disorder, compared with 70% of age-associated markers (Experimental Procedures, Figure S5). Furthermore, whereas age-associated markers tended to have a low methylation fraction that increased with age, HIV-associated markers were more equally balanced between low and high methylation states (Figure 4C)."
In summary, we have shown that an extrinsic perturbation to a human population, driven by HIV infection and cART, is capable of inducing changes in the epigenomic state of affected individuals. This perturbation may influence regulation of HLA gene expression and also encompasses signatures of aging. Our findings help address a long-standing debate regarding the effects of HIV infection on biological aging in cART-treated individuals, in a manner that can be assessed numerically using an epigenome-based readout. Taken together, our findings show that the epigenome adds a quantitative means of assessing the interaction of HIV with normal and pathogenic processes associated with aging, and they shed light on the underlying mechanisms by which acute and chronic viral infection impact the host.
We have shown that methylome-wide changes previously ascribed to aging are also induced by HIV (Figures 1 and 3). By using highly accurate, externally trained and validated models of biological aging, our study provides a robust estimate of a 5-year age advancement in HIV/cART individuals (Figure 2). These results, in combination with the link between molecular age advancement and increased mortality risk (Marioni et al., 2015), support the idea that chronic HIV infection is accompanied by a tangible gerontological phenotype. In addition to an aggregate estimate of HIV age advancement, the methylation aging model allows for patient-by-patient estimates. Patients deemed more likely to suffer from HIV-mediated aging effects might be placed on alterative schedules for preventative care, including early screening and further testing if warranted.
"we find that both chronic and recent HIV infection lead to an average aging advancement of 4.9 years, increasing expected mortality risk by 19%.....age advancement occurs early in the course of disease as a consequence of acute infection or reaction to drug treatment.....patients more recently infected with HIV (<5 years) had no significant difference in age advancement from those patients with chronic (>12 years) infection (p > 0.5, Mann-Whitney U Test; Figure 2F). Similar findings emerged from a regression analysis of the chronological versus biological time since infection: the slope did not differ from one (0.98 ± 0.06, SE) whereas the y-intersect was significantly positive (5.2 ± 0.9; Figures 2E and 2F). These findings lend support to the theory that age advancement occurs early in the course of disease as a consequence of acute infection or reaction to drug treatment.....For uninfected controls, the calculated biological age had a very high concordance with chronological age (Figure 2D, Pearson's r = 0.94). In contrast, the HIV+ patients had a biological age advancement of 4.9 years on average (p < 10-8 by Student's t test, 95% confidence interval 3.4-7.1 years, Figures 2E and 2F)......For uninfected controls, the calculated biological age had a very high concordance with chronological age (Figure 2D, Pearson's r = 0.94). In contrast, the HIV+ patients had a biological age advancement of 4.9 years on average (p < 10-8 by Student's t test, 95% confidence interval 3.4-7.1 years, Figures 2E and 2F). These results were consistent with our previous unsupervised analysis (Figures 1B and 1C) in suggesting that HIV infection leads to advanced aging.....These findings reinforce previous reports that PRC2 targets are irreversibly repressed by methylation during the aging process (Beerman et al., 2013, Deaton and Bird, 2011, Teschendorff et al., 2010). Interestingly, markers associated with HIV but not aging had a very different functional enrichment profile (Figure 4B), indicating an additional mechanism(s) for epigenetic alteration associated with HIV."
"Age Advancement Is Independent of HIV Duration
Notably, patients more recently infected with HIV (<5 years) had no significant difference in age advancement from those patients with chronic (>12 years) infection (p > 0.5, Mann-Whitney U Test; Figure 2F). Similar findings emerged from a regression analysis of the chronological versus biological time since infection: the slope did not differ from one (0.98 ± 0.06, SE) whereas the y-intersect was significantly positive (5.2 ± 0.9; Figures 2E and 2F). These findings lend support to the theory that age advancement occurs early in the course of disease as a consequence of acute infection or reaction to drug treatment (Guaraldi et al., 2011, Smith et al., 2012). The lack of an increase of age advancement with disease duration seems to contradict alternative views that HIV-mediated aging occurs through cumulative effects of latent virus (Appay and Rowland-Jones, 2002) or chronic therapeutic intervention (Torres and Lewis, 2014). We did however observe less variation in age advancement within the chronically infected HIV+ individuals (Figure 2F, p < 0.002, Bartlett's test relative to recently infected group), perhaps reflecting the comparative stability of infection and immune response on long-term cART therapy (Luz et al., 2014, Rosenblatt et al., 2005).
cell-type-associated changes could be responsible for the observed increase in biological age in the HIV+ cases. To assess this possibility, we constructed a multivariate linear model in which cell type composition variables and HIV status were used to predict biological age as measured by the methylome (Table 1). In this model, the presence of HIV was associated with an age advancement of 3.8 ± 1.1 years, while the presence of natural killer cells accounted for additional increases in biological age. In an even more conservative test, we modeled age advancement with cell type composition variables alone and found that the unexplained variation in this model still had a significant association with HIV infection (p = 0.02, Likelihood Ratio Test, Table 1). Thus, even in a very conservative analysis, HIV infection has association with advanced aging that is independent of cell composition.
Analysis of the whole-blood data identified a single genomic region that was enriched in CpG markers associated with HIV; this region, consisting of 10 Mb on chromosome 6 including histone gene cluster 1 and the entire HLA locus, had particularly reduced methylation levels in HIV+ cases as compared to HIV- controls (p < 10-10, Figure 5A; Experimental Procedures). HLA genes encode the Major Histocompatibility Complexes (MHCs), the key antigen-presenting molecules that govern the acquired immune response and impact innate immunity (Figure 5B) (Goulder and Walker, 2012). We found that the differentially methylated markers surround the rs2395029 variant, for which common genetic variation has been repeatedly implicated in HIV host control (Figures 5C and 5D) (Fellay et al., 2007, International HIV Controllers Study et al., 2010). Examination of this locus in the validation samples of purified neutrophils and CD4+ T cells identified the HCP5 gene body as particularly differentially methylated in neutrophils (Figures 5E, 5F, and S6). As further evidence that the observed changes are functional, we found that the amount of methylation at this gene was correlated with a patient's CD4+/CD8+ T cell ratio (Figure S6). Taken together, these results indicate that the HLA locus is likely differentially methylated across blood cell types and also changes within individual cell types in response to HIV. An intriguing interpretation of our results is that some of the previously reported changes in HLA expression and corresponding HIV control (Apps et al., 2013) are attributable to methylation dynamics."
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Methylome-wide Analysis of Chronic HIV Infection Reveals Five-Year Increase in Biological Age and Epigenetic Targeting of HLA
Highlights
• •Methylome-wide analysis of HIV chronically infected, cART treated individuals
• •HIV+ individuals have an epigenetic age 4.9 years older than healthy controls
• •HLA locus is hypomethylated in HIV+ individuals
• •HIV methylation aging signature is validated in purified cells
Summary
HIV-infected individuals are living longer on antiretroviral therapy, but many patients display signs that in some ways resemble premature aging. To investigate and quantify the impact of chronic HIV infection on aging, we report a global analysis of the whole-blood DNA methylomes of 137 HIV+ individuals under sustained therapy along with 44 matched HIV- individuals. First, we develop and validate epigenetic models of aging that are independent of blood cell composition. Using these models, we find that both chronic and recent HIV infection lead to an average aging advancement of 4.9 years, increasing expected mortality risk by 19%. In addition, sustained infection results in global deregulation of the methylome across >80,000 CpGs and specific hypomethylation of the region encoding the human leukocyte antigen locus (HLA). We find that decreased HLA methylation is predictive of lower CD4 / CD8 T cell ratio, linking molecular aging, epigenetic regulation, and disease progression.
Introduction
It is an open question why some people show early or delayed onset of aging-associated disorders (Kennedy et al., 2014). Recent studies have found that aging is associated with epigenetic changes (Christensen et al., 2009, Day et al., 2013, Heyn et al., 2012, Numata et al., 2012, West et al., 2013), and based on this work we (Hannum et al., 2012) and others (Horvath, 2013, Weidner et al., 2014) have built models capable of predicting a person's age using DNA methylation patterns across a large number of CpG sites. Although these models are fairly accurate, errors of prediction-differences between the chronological and predicted age-serve as a quantitative readout of the relative advancement or retardation of the "biological age" of an individual. Biological age advancement has been correlated with factors such as gender, genetic polymorphisms, and diseases including cancer and diabetes, and it may influence the onset of other age-associated disorders (Day et al., 2013, Hannum et al., 2012). A recent longitudinal study validated the clinical utility of these models by demonstrating a link between biological age advancement and increased mortality rates (Marioni et al., 2015).
Biological aging has become of particular interest in treatment of HIV, in which the development of combination Anti-Retroviral Therapy (cART) now enables infected individuals to live many decades (Deeks, 2011, Deeks et al., 2013, Maartens et al., 2014). Several studies have suggested links between chronic HIV infection and early onset of neurodegeneration (Nightingale et al., 2014), liver or kidney failure (Joshi et al., 2011, Kovari et al., 2013), cancer (Dubrow et al., 2012), cardiovascular disease (Freiberg et al., 2013), and telomere shortening (Leeansyah et al., 2013, Pathai et al., 2013), leading to the hypothesis that HIV+ patients might experience advanced or accelerated aging (Appay and Rowland-Jones, 2002, Guaraldi et al., 2011, Smith et al., 2012). While these studies report rough estimates of HIV-mediated age advancement in the range of 0-20 years, it has been difficult to accurately quantify this number due to sampling effects, co-morbidities, and relatively low incidence rates of any single age-associated disease. To this effect, the existence, extent, and molecular basis of a bona-fide increase in aging have been unclear (Althoff et al., 2014, Solomon et al., 2014), in part due to lack of an objective biological clock or aging biomarker.
In parallel with such epidemiological observations, a number of studies report age effects using blood-based biomarkers. Analysis of cell surface markers in T cells has shown HIV+ subjects to show phenotypes of older cells (Cao et al., 2009). Other studies have observed shortened telomeres in certain cell populations (Rickabaugh et al., 2011) as well as whole blood (Zanet et al., 2014), indirectly linking HIV to aging via the well-studied connection between telomere length and age (Lindsey et al., 1991, Cawthon et al., 2003). Furthermore, a recent analysis of untreated HIV+ individuals found DNA methylation sites that are associated with both HIV infection and age (Rickabaugh et al., 2015). Together, these results raise the possibility that HIV infection results in an increase in biological age. Many questions remain, however: Are the epigenetic changes associated with HIV the same as those previously identified (Hannum et al., 2012, Horvath, 2013) in normal individuals as markers of "biological age," and how complete is the correspondence between these two responses? What is the quantitative effect on aging in years, and is it fixed age advancement or continuous acceleration? What is the impact on aging of chronic HIV infection and sustained cART treatment? Are there other impacts of HIV on the methylome that are unrelated to aging?
Here we begin to address these questions by analyzing the methylomes of HIV-infected, cART-treated subjects, in which we observe a strong shared phenotype of HIV and age. To understand this signal, we develop models of biological age that allow us to establish a clear quantitative link between HIV infection and aging as observed in the general population. We identify both global and targeted epigenomic effects of HIV, including specific hypomethylation of the HLA locus. Together, these results shed light on the epigenetic consequences and/or gerontological aspects of chronic HIV infection.
Results
Genome-wide DNA Methylation Profiling
To determine whether HIV is associated with signs of aberrant biological aging, samples of whole-blood DNA were obtained from 137 HIV-infected, cART-treated but otherwise healthy non-Hispanic white males (no hepatitis C co-infection, no diabetes, and high adherence to therapy) and 44 healthy non-Hispanic white male controls (Table S1; Figure S1). Genome-wide methylation profiles of each sample were determined using the Illumina Infinium HumanMethylation450 BeadChip array. Data were normalized and controlled for quality using standard techniques, resulting in removal of two control patients due to poor signal (Experimental Procedures).
Unsupervised Analysis Shows Shared Phenotypes of HIV and Age
As a preliminary exploration of this dataset, we ran an unsupervised analysis to identify age-associated methylation sites and their relation to HIV infection. Analysis of a previous methylome-wide screen of 538 healthy subjects (Hannum et al., 2012) identified as many as 61,592 methylation sites associated with age at a 1% false-discovery rate (FDR) (likelihood ratio test in multivariate regression model with Benjamini-Hochberg correction). Validation of these sites in whole blood from a second control cohort from the European Prospective Investigation into Cancer and Nutrition (Riboli et al., 2002) (EPIC, N = 662) confirmed 26,927 of these sites as strongly associated with age (Figure 1A; Table S2).
Among these validated age-associated sites, we found a striking association with methylation in the HIV+ patients relative to healthy controls (p < 10-100, Figure 1B). Further analysis of these sites found a positive association of the first principal component with both age and HIV status (Figure 1C; Table S3, association by multivariate linear model p < 10-8). These findings support a link between HIV infection and aging (Rickabaugh et al., 2015), as quantitatively measured by epigenomic profiling (Figure 1D).
Benchmarking and Refinement of Epigenetic Aging Models
Given the shared effects of HIV and aging, we sought to determine whether HIV causes the same biological aging signature as previously found in cohorts of uninfected individuals (Hannum et al., 2012, Horvath, 2013, Marioni et al., 2015). We tested aging models from both our group (Hannum et al., 2012) and Horvath (Horvath, 2013) in independent datasets derived from whole blood samples (Hannum et al., 2012, Riboli et al., 2002; Table S4). Although the Hannum and Horvath modeling efforts were based on different methodologies and training data, we found they made very similar predictions (r = 0.9, Pearson's correlation, Figure 2A) and furthermore that a consensus of the two models outperformed either model individually (Figures 2B and 2C; Table S4). For this reason, we used this consensus model for all remaining analyses.
A potential issue with these models arises in the fact that methylation profiles from whole blood are influenced by cell composition, and different cell types have different methylation states (Jaffe and Irizarry, 2014). These differences might be particularly pronounced in HIV-infected patients, some of whom have low CD4+ T cell counts (Trono et al., 2010). To understand the sensitivity of epigenetic aging models to cell type composition, we downloaded two datasets profiling sorted cells across shared sets of individuals (Absher et al., 2013, GEO: GSE59250; Reynolds et al., 2014, GEO: GSE56046). Among these sorted cell datasets, we saw good concordance of epigenetic age predictions with chronological age (Figures S2A-S2F). Epigenetic age was reproducible across different cell types profiled from the same patients, with high agreement of age estimates (r >0.77-0.88) and moderate but very significant agreement of age advancement (Pearson's r > 0.45-0.68; p < 0.0001 for all associations, Figures S2G-S2J).
While we therefore expect the contribution of cell composition to be minimal, we nonetheless developed an algorithm to individually normalize each methylation profile using methylation-derived cell type information. In brief, we used a previously reported method (Jaffe and Irizarry, 2014) to reliably predict blood composition (Figure S3) and adjust out the expected contribution of cell-type-specific effects. This procedure greatly limited the effects of age- and HIV-induced blood composition changes in downstream analyses (Experimental Procedures; Figure S4).
HIV+ Individuals Have Advanced DNA Methylation Age
We next used this consensus aging model to calculate the "biological age" of each individual in our cohort (Table S4). For uninfected controls, the calculated biological age had a very high concordance with chronological age (Figure 2D, Pearson's r = 0.94). In contrast, the HIV+ patients had a biological age advancement of 4.9 years on average (p < 10-8 by Student's t test, 95% confidence interval 3.4-7.1 years, Figures 2E and 2F). These results were consistent with our previous unsupervised analysis (Figures 1B and 1C) in suggesting that HIV infection leads to advanced aging. Furthermore, we found that the age advancement of HIV+ individuals was negatively correlated with the ratio of CD4+ / CD8+ T lymphocytes (Spearman's rho = -0.2, p < 0.02). CD4+ T cells are a major indicator of immune integrity (Leung et al., 2013, Serrano-Villar et al., 2014) and are inversely associated with morbidity and mortality, including from non-AIDS defining diseases (El-Sadr et al., 2006); similarly, the CD4/CD8 ratio predicts non-AIDS morbidity (Leung et al., 2013, Serrano-Villar et al., 2014). This finding links biological aging of HIV-infected individuals to a clinical measure of disease progression, and it raises the possibility that patients with stable immune responses may be less affected by the advanced aging phenotype. Taking into account a recently estimated 4.2% increase in mortality risk per year of biological age advancement using the Hannum model (Marioni et al., 2015), the changes observed in HIV+ patients result in an expected total mortality risk increase of 19%.
Age Advancement Is Independent of HIV Duration
Notably, patients more recently infected with HIV (<5 years) had no significant difference in age advancement from those patients with chronic (>12 years) infection (p > 0.5, Mann-Whitney U Test; Figure 2F). Similar findings emerged from a regression analysis of the chronological versus biological time since infection: the slope did not differ from one (0.98 ± 0.06, SE) whereas the y-intersect was significantly positive (5.2 ± 0.9; Figures 2E and 2F). These findings lend support to the theory that age advancement occurs early in the course of disease as a consequence of acute infection or reaction to drug treatment (Guaraldi et al., 2011, Smith et al., 2012). The lack of an increase of age advancement with disease duration seems to contradict alternative views that HIV-mediated aging occurs through cumulative effects of latent virus (Appay and Rowland-Jones, 2002) or chronic therapeutic intervention (Torres and Lewis, 2014). We did however observe less variation in age advancement within the chronically infected HIV+ individuals (Figure 2F, p < 0.002, Bartlett's test relative to recently infected group), perhaps reflecting the comparative stability of infection and immune response on long-term cART therapy (Luz et al., 2014, Rosenblatt et al., 2005).
Age Advancement Is Independent of Cellular Composition
While the direct effects of cell type composition on the whole-blood methylome were corrected by the adjustment described above (also see Experimental Procedures), we considered that it was still possible that changes in cell type composition could lead to downstream, indirect changes in the epigenomes of all blood cells. If this were the case, cell-type-associated changes could be responsible for the observed increase in biological age in the HIV+ cases. To assess this possibility, we constructed a multivariate linear model in which cell type composition variables and HIV status were used to predict biological age as measured by the methylome (Table 1). In this model, the presence of HIV was associated with an age advancement of 3.8 ± 1.1 years, while the presence of natural killer cells accounted for additional increases in biological age. In an even more conservative test, we modeled age advancement with cell type composition variables alone and found that the unexplained variation in this model still had a significant association with HIV infection (p = 0.02, Likelihood Ratio Test, Table 1). Thus, even in a very conservative analysis, HIV infection has association with advanced aging that is independent of cell composition.
We also sought to experimentally assess if the observed age advancement due to HIV infection was observed in purified cell populations. Using standard calculations of statistical power, we estimated that a sample of 48 patients, balanced approximately between cases and controls, would have 81% power to detect the same aging advancement effect as our primary screen at p < 0.01. Accordingly, this number of subjects was prospectively recruited from the University of Nebraska Medical Center under an approved IRB protocol (501-15-EP), and blood obtained following informed consent (Experimental Procedures; Table S5). Whole blood was separated immunomagnetically to isolate pure populations of neutrophils and CD4+ T cells.
As in whole blood, unsupervised analysis showed a clear effect of HIV in age-associated methylation markers (Figures 3A and 3B ). Application of epigenetic models of aging in these pure-cell datasets showed good concordance of predicted age with chronological age in both cell types (Figures 3C-3F). In neutrophils, the Hannum model predicted a 2.5 year increase in age due to HIV infection (p < 0.03, 95% CI 0.6-5.0 years, Figure 3E) whereas the Horvath model showed a smaller effect of 0.4 year (p > 0.05). In contrast, CD4+ T cells had a much stronger and more consistent HIV response in both models, with the consensus aging model showing an increase of 5.7 years in the HIV+ subjects (p < 10-5, 95% CI 3.4-7.9 years, Figure 3F). These data indicate that the effect of epigenetic age advancement is not merely an artifact of changing blood composition, but likely reflects true aging signals. The stronger effect size within CD4+ T cells (Figures 3G and 3H) suggests that these cells may be exposed to more age-like stress than neutrophils, although further work is needed to understand how disease may affect aging rates across different cell types and tissues.
HIV and Aging Have Shared and Distinct Methylation Patterns
Having identified a large effect of HIV infection on age associated methylation signals, we then sought to better understand the wider changes instigated by HIV. We identified 81,361 CpG markers associated with HIV infection (Benjamini-Hochberg corrected p < 0.01; likelihood ratio test using a multivariate linear model, Table S2). Of these, 2,569 upregulated markers and 1,769 downregulated markers were also associated with aging, a 3.2 and 1.4-fold enrichment over random expectation, respectively (Figure 4A, Fisher's Exact Test p < 10-30, Table S6). We found that markers associated with both HIV and aging were enriched in DNase hypersensitivity sites and CpG islands, suggesting methylation changes in DNA regions under active regulation. These CpG markers were also enriched in binding sites for polycomb repressive complex (PRC2) (Figure 4B), a switch that tightly regulates genes required for differentiation and renewal, and in Drosophilia is linked to longevity (Siebold et al., 2010). These findings reinforce previous reports that PRC2 targets are irreversibly repressed by methylation during the aging process (Beerman et al., 2013, Deaton and Bird, 2011, Teschendorff et al., 2010). Interestingly, markers associated with HIV but not aging had a very different functional enrichment profile (Figure 4B), indicating an additional mechanism(s) for epigenetic alteration associated with HIV.
We have previously reported that age-associated markers in older subjects tend away from a fully methylated or unmethylated state and instead move toward disorder (with a methylation fraction of 50% representing complete disorder) (Hannum et al., 2012). We found that HIV-infected patients displayed a similar trait: among markers associated with HIV, 66% tended toward disorder, compared with 70% of age-associated markers (Experimental Procedures, Figure S5). Furthermore, whereas age-associated markers tended to have a low methylation fraction that increased with age, HIV-associated markers were more equally balanced between low and high methylation states (Figure 4C).
HIV Is Associated with Hypomethylation of the HLA Locus, Independent of Aging
Thus far, we had observed multiple effects of HIV on the methylome, including changes in cellular composition, age advancement, and a general increase in methylome disorder. We next sought to determine whether there are specific genomic regions for which the methylation state is particularly associated with HIV infection, independent of aging or other factors (Experimental Procedures). Toward this aim, we conducted an analysis of HIV-associated CpG markers independent of disorder or age, controlling for the effects of cellular composition. Analysis of the whole-blood data identified a single genomic region that was enriched in CpG markers associated with HIV; this region, consisting of 10 Mb on chromosome 6 including histone gene cluster 1 and the entire HLA locus, had particularly reduced methylation levels in HIV+ cases as compared to HIV- controls (p < 10-10, Figure 5A; Experimental Procedures). HLA genes encode the Major Histocompatibility Complexes (MHCs), the key antigen-presenting molecules that govern the acquired immune response and impact innate immunity (Figure 5B) (Goulder and Walker, 2012). We found that the differentially methylated markers surround the rs2395029 variant, for which common genetic variation has been repeatedly implicated in HIV host control (Figures 5C and 5D) (Fellay et al., 2007, International HIV Controllers Study et al., 2010). Examination of this locus in the validation samples of purified neutrophils and CD4+ T cells identified the HCP5 gene body as particularly differentially methylated in neutrophils (Figures 5E, 5F, and S6). As further evidence that the observed changes are functional, we found that the amount of methylation at this gene was correlated with a patient's CD4+/CD8+ T cell ratio (Figure S6). Taken together, these results indicate that the HLA locus is likely differentially methylated across blood cell types and also changes within individual cell types in response to HIV. An intriguing interpretation of our results is that some of the previously reported changes in HLA expression and corresponding HIV control (Apps et al., 2013) are attributable to methylation dynamics.
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