Epigenetic signature of human immune aging in the GESTALT study
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eLife assessment
This is an important study reanalysing previously published datasets to understand methylation changes during aging. The evidence supporting the conclusions is solid and sheds new light on features of aging in cells, highlighting the concept of cell-specific methylation changes and their relationship to other physiological changes such as inflammation that may impact methylation patterns. This work will be of broad interest to cell and molecular biologists.
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Abstract
Age-associated DNA methylation in blood cells convey information on health status. However, the mechanisms that drive these changes in circulating cells and their relationships to gene regulation are unknown. We identified age-associated DNA methylation sites in six purified blood-borne immune cell types (naive B, naive CD4 + and CD8 + T cells, granulocytes, monocytes, and NK cells) collected from healthy individuals interspersed over a wide age range. Of the thousands of age-associated sites, only 350 sites were differentially methylated in the same direction in all cell types and validated in an independent longitudinal cohort. Genes close to age-associated hypomethylated sites were enriched for collagen biosynthesis and complement cascade pathways, while genes close to hypermethylated sites mapped to neuronal pathways. In silico analyses showed that in most cell types, the age-associated hypo- and hypermethylated sites were enriched for ARNT (HIF1β) and REST transcription factor (TF) motifs, respectively, which are both master regulators of hypoxia response. To conclude, despite spatial heterogeneity, there is a commonality in the putative regulatory role with respect to TF motifs and histone modifications at and around these sites. These features suggest that DNA methylation changes in healthy aging may be adaptive responses to fluctuations of oxygen availability.
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Author Response
Reviewer #1 (Public Review):
This research aimed to discern the pattern of methylation changes that occur during aging, distinguishing between a unified specific mechanism and stochastic changes. To date, no unified hypothesis exists to guide our understanding of the changes in chromatin geography observed during the aging of cells. This work analysed six different types of purified blood-borne white blood cells allowing comparison across different immune cell subsets to determine if similar patterns occurred in all cell populations. Intriguingly, each subset exhibited its own distinct differential methylation rather than a single program. However, a core set of gene changes close to age-associated CpGs was identified suggesting that a central program existed, but that individual cell type function and metabolism …
Author Response
Reviewer #1 (Public Review):
This research aimed to discern the pattern of methylation changes that occur during aging, distinguishing between a unified specific mechanism and stochastic changes. To date, no unified hypothesis exists to guide our understanding of the changes in chromatin geography observed during the aging of cells. This work analysed six different types of purified blood-borne white blood cells allowing comparison across different immune cell subsets to determine if similar patterns occurred in all cell populations. Intriguingly, each subset exhibited its own distinct differential methylation rather than a single program. However, a core set of gene changes close to age-associated CpGs was identified suggesting that a central program existed, but that individual cell type function and metabolism shaped the overall chromatin landscape for the population. These findings establish a new framework for considering the aging process and open new questions about how the individual clocks of different populations might be regulated. While circulating cells are readily accessible for evaluation in humans, the majority of immune cells that regulate immune homeostasis are found within the tissues of the body. Whether these cells exhibit a similar profile to circulating cells or are rather shaped by their tissue or organ-specific ecosystem remains to be determined. In this setting, these tissue-resident cells are exposed to very different oxygen tensions and metabolic substrates. Furthermore, genes identified have been associated with aging, they concurrently appear to be associated with inflammation, thus it is not clear whether aging and low-grade inflammation are inherently linked, or whether these two pathways can be segregated. Thus a number of questions remain warranting further investigation.
The reviewer makes a very good point regarding different tissue resident cells being exposed to different oxygen and metabolic stress. In the reviewed manuscript we have Arid3a coming up as one of the transcription factors with motifs in and around probes hypermethylated with age in monocytes. Arid3a is known to target inflammatory genes but future research is warranted to implicate the link between aging and low-grade inflammation. To address the comment about connection between aging and low-grade inflammation, in the revised manuscript, we have incorporated new analysis by looking into SomaScan array derived protein levels of seven cytokines from the same cohort of donors. We tested the hypothesis that part of the age-associated changes in DNA methylation are connected with the well-known age-related proinflammatory state. We have now added the details in the Results and Methods sections. Briefly, we run two regression models (CpGi~age+sex and CpGi~age+sex+analytej, where i is each CpG probe from EPIC array and j is each of the seven cytokines). We find that change in DNA methylation levels in nearly 70009000 CpG sites in CD4 cells and 124 CpG sites in B cells that were originally age-associated, also are associated with increasing levels of TNFRSF1A, TNFRSF1B and TNF-alpha levels thereby indicating a link between DNA methylation change and aging as well as inflammatory cytokines levels.
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eLife assessment
This is an important study reanalysing previously published datasets to understand methylation changes during aging. The evidence supporting the conclusions is solid and sheds new light on features of aging in cells, highlighting the concept of cell-specific methylation changes and their relationship to other physiological changes such as inflammation that may impact methylation patterns. This work will be of broad interest to cell and molecular biologists.
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Reviewer #1 (Public Review):
This research aimed to discern the pattern of methylation changes that occur during aging, distinguishing between a unified specific mechanism and stochastic changes. To date, no unified hypothesis exists to guide our understanding of the changes in chromatin geography observed during the aging of cells. This work analysed six different types of purified blood-borne white blood cells allowing comparison across different immune cell subsets to determine if similar patterns occurred in all cell populations. Intriguingly, each subset exhibited its own distinct differential methylation rather than a single program. However, a core set of gene changes close to age-associated CpGs was identified suggesting that a central program existed, but that individual cell type function and metabolism shaped the overall …
Reviewer #1 (Public Review):
This research aimed to discern the pattern of methylation changes that occur during aging, distinguishing between a unified specific mechanism and stochastic changes. To date, no unified hypothesis exists to guide our understanding of the changes in chromatin geography observed during the aging of cells. This work analysed six different types of purified blood-borne white blood cells allowing comparison across different immune cell subsets to determine if similar patterns occurred in all cell populations. Intriguingly, each subset exhibited its own distinct differential methylation rather than a single program. However, a core set of gene changes close to age-associated CpGs was identified suggesting that a central program existed, but that individual cell type function and metabolism shaped the overall chromatin landscape for the population. These findings establish a new framework for considering the aging process and open new questions about how the individual clocks of different populations might be regulated. While circulating cells are readily accessible for evaluation in humans, the majority of immune cells that regulate immune homeostasis are found within the tissues of the body. Whether these cells exhibit a similar profile to circulating cells or are rather shaped by their tissue or organ-specific ecosystem remains to be determined. In this setting, these tissue-resident cells are exposed to very different oxygen tensions and metabolic substrates. Furthermore, genes identified have been associated with aging, they concurrently appear to be associated with inflammation, thus it is not clear whether aging and low-grade inflammation are inherently linked, or whether these two pathways can be segregated. Thus a number of questions remain warranting further investigation.
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Reviewer #2 (Public Review):
The authors utilized publicly available datasets to investigate age-related DNA methylation changes in six immune cell types. They identified 350 differentially methylated sites that were changing in the same directions among all cell types, while most of the differentially methylated sites were cell type-specific during aging. Further analyses of enriched pathways and motifs indicate that these DNA methylation changes may be induced by the fluctuations in oxygen availability.
Analyzing cell type-specific DNA methylation data and comparing cross-sectional and longitudinal datasets, the authors are able to identify age-associated DNA methylation sites that may be regulated by a common mechanism in aging. However, sex differences should be considered, and the proposed mechanism could spur future studies to …
Reviewer #2 (Public Review):
The authors utilized publicly available datasets to investigate age-related DNA methylation changes in six immune cell types. They identified 350 differentially methylated sites that were changing in the same directions among all cell types, while most of the differentially methylated sites were cell type-specific during aging. Further analyses of enriched pathways and motifs indicate that these DNA methylation changes may be induced by the fluctuations in oxygen availability.
Analyzing cell type-specific DNA methylation data and comparing cross-sectional and longitudinal datasets, the authors are able to identify age-associated DNA methylation sites that may be regulated by a common mechanism in aging. However, sex differences should be considered, and the proposed mechanism could spur future studies to test it.
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Reviewer #3 (Public Review):
In this study, titled "Epigenetic signature of human immune aging: the GESTALT study," the authors reanalysed data from five highly purified human immune cell types from 55 healthy volunteers across a wide age range to characterize age-related changes in DNA methylation status. Additionally, they performed some integrative analyses with chromatin state and transcriptional data. Findings support that age-related DNA methylation changes are predominantly cell type-specific. Out of thousands of age-associated sites, only 350 sites were differentially methylated in the same direction in all cell types and validated in an independent longitudinal cohort. Some conserved changes exist, which appear to be underpinned by alterations in the hypoxia response, with linked enrichment of transcription factor binding …
Reviewer #3 (Public Review):
In this study, titled "Epigenetic signature of human immune aging: the GESTALT study," the authors reanalysed data from five highly purified human immune cell types from 55 healthy volunteers across a wide age range to characterize age-related changes in DNA methylation status. Additionally, they performed some integrative analyses with chromatin state and transcriptional data. Findings support that age-related DNA methylation changes are predominantly cell type-specific. Out of thousands of age-associated sites, only 350 sites were differentially methylated in the same direction in all cell types and validated in an independent longitudinal cohort. Some conserved changes exist, which appear to be underpinned by alterations in the hypoxia response, with linked enrichment of transcription factor binding motifs related to ARNT and REST. The authors conclude that DNA methylation changes in healthy aging may represent adaptive responses to fluctuations in oxygen availability.
Strengths:
- The study utilised data from a large cohort of individuals (n=55), with participants ranging in age from their 20s to 80s, providing a comprehensive age-related analysis.
- The data set reanalysed was based on highly purified cells rather than unfractionated PBMCs. This revealed the largely cell-type-specific nature of these changes and demonstrated that conflicting directional changes can cancel each other out, going undetected at the PBMC level.
- The authors were able to verify the DNA methylation changes that were conserved across cell types longitudinally in PBMCs by reanalyzing published datasets from the InCHIANTI study, adding robustness to their findings.
Weaknesses:
- The authors make statements in the abstract and manuscript that overreach the study's findings. Specifically, they claim that "DNA methylation changes in healthy aging may represent adaptive responses to fluctuations in oxygen availability." In reality, the study shows that a small minority of conserved DNA methylation changes across hematopoietic cell types appear to be driven by hypoxia response processes. The study does not demonstrate that hypoxia response processes account for a large proportion of DNA methylation changes or that these processes apply to non-hematopoietic cell types. These statements should be put into context relative to the study findings.
- The authors should make it clear in the introduction and methods section that this current study is merely reanalysing a data set they published before. Also, the authors should describe in the introduction the key findings of the initial analysis as presented in their 2021 Immunity publication.
- In some instances, the manuscript lacks citations to support claims.
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