Longitudinal Multi-Omic Study of Aging Determinants

Longitudinal Multi-Omic Study of Aging Determinants

Prof. Maxim N. Artyomov – The Artyomov Lab, Department of Pathology & Immunology, Washington University School of Medicine in St. Louis, USA

Simply Put

Human studies are challenging because of the variability inherent to human lifestyles, genetics, etc. Aging studies are challenging because of the multi-factorial nature of the aging process.

One way to minimize these issues is to use as restricted and homogeneous cohorts as possible and to profile on them as many aging features as we can. By means of unbiased multidimensional cutting-edge profiling techniques we can study aging spanning scales from a single-cell to purified cell types to blood cell mixtures and physiological/clinical blood testing parameters. The following studies aim to establish the baseline in understanding healthy human aging, focusing on cellular and soluble determinants of human blood.

Understanding Omic Signatures of Healthy Aging in Purified Cell Type (Pilot Study)

Many studies investigate blood composition changes with age in bulk. This described project aims to understand the multi-omic cellular and soluble determinants on the individual cell basis rather than whole PBMC. The research’s focus was to characterize epigenomic, transcriptomic, and proteomic alterations in purified CD14+CD16- monocytes, as well as the corresponding proteomic and metabolomic changes in plasma.

In the pilot phase of this project we compared monocytes from 20 young and 20 old individuals. Given that aging depends on multiple genetic and environmental factors, the pilot cohort was selected to be as homogenous as possible in these factors. That would help establish baseline aging determinants and evaluate the statistical power required for each type of change (e.g., transcriptomic vs. epigenetic). Specifically, the pilot cohort was restricted to non-smocking Caucasian males with a healthy body-mass ratio (BMI<30) and self-reported absence of underlying inflammatory conditions, acute viral infections, and cancer. This pilot’s results will inform the study design for the larger cohorts’ characterization that would study healthy aging in diverse sex and race backgrounds.

The multi-omics characterization included enhanced Reduced Representation Bisulfite Sequencing (eRRBS), Ultra-Low-Input ChIP-Sequencing (ULI-ChIP-seq) of 5 key chromatin marks, RNA-sequencing, and proteomic profiling of the isolated monocytes. Additionally, the plasma of the participants was characerized in terms of plasma proteins (via SomaLogic), unbiased metabolic profiling and cytokine levels.

Pilot Phase Results

For the first time, the study has established a set of continuous DNA regions that change methylation levels with age in a primary human cell type, which provides potential cues to the aging biology fundamentals. Integrative analysis revealed a number of the transcription factor binding sites that can regulate DNA methylation levels with age and DNA polymorphisms that can directly affect the immune system’s fitness. It also demonstrated the remarkable stability of the classical monocytes’ chromatin and transcriptional signatures in human aging.

The pilot phase of this project is accepted for publication in the first volume of Nature Aging. The preprint can be already viewed HERE

Easily accessible visualization and exploration of all data from this study and detailed descriptions of protocols and computational pipelines are available ONLINE

Establishing Cross-sectional and Short-term Longitudinal Trajectories of Healthy Aging (Extended Pilot)

This project extends the previous pilot study to characterize a broader set of blood determinants of healthy aging across multiple ages, including short-term 2-year follow-up sampling.

The data include targeted characteristics such as

  • blood differential,
  • a set of clinical blood tests (~30),
  • broad profiling of plasma proteomics and metabolomics,
  • characterization of the blood composition using CYTOF and single-cell RNA-seq profiling,
  • continued profiling of isolated monocytes using RNA-sequencing,
  • and whole genome bisulfite sequencing.

Following the principles outlined in the first pilot study, this extended pilot study annually collects the blood from male and female donors between ages 25 and 80 over the three consecutive years 2018/2019 to 2020/2021. All donors are restricted to be Caucasian non-smokers, with a healthy body-mass ratio (BMI < 30) and self-reported absence of underlying inflammatory conditions, acute viral infections, and cancer. These extended pilot project’s results will inform the study design for the larger cohorts’ characterization that would study healthy aging in diverse backgrounds.

General scheme of the cross-sectional and short-term longitudinal study profiling is presented on the figure below.

One of the essential lessons from the Pilot Phase study comparing monocytes of old and young patients was that plasma measurements help distinguish between different ages. Accordingly, the profiling of the plasma metabolic levels, cytokines, 1100 plasma proteins, as well as the new lipidomic profiling of plasma samples, will be performed.

The second important lesson from the Pilot Phase was that the blood composition could be changing significantly with age. Accordingly, the profiling techniques that allow capturing the state of the complex issues at the single-cell resolution will be employed, such as single-cell RNA-seq for transcriptional states and CyTOF for total levels of individual chromatin marks.

In this context, single-cell RNA-seq technology allows simultaneous capturing transcriptional states of all cell types present in the blood – from monocytes to various kinds of T-cells and NK-cells – and establishing which cell types are most sensitive to aging from the transcriptional point of view.

Moreover, modern 10x single-cell RNA-seq provides not only transcriptional but also TCR-repertoire characterization on a different level. TCR repertoire is one of the major determinants of aging on the cellular level, as with age, new T-cells’ production decreases dramatically due to the age-related decrease in the thymus’ function. The number of T-cells decreases in the blood, and this decrease is accompanied by an even more striking clonal reduction in the peripheral T-cell receptor repertoire.

Single-cell RNA-seq profiling, including TCR-repertoire on collected peripheral blood mononuclear cells (PBMC), will provide a comprehensive characterization of major changes between young and old blood subtypes. That fact, in turn, will allow direct evaluation if any cellular subpopulations are changing during aging, or, inversely, whether the blood changes are completely driven by the changes in frequencies of individual cell types.

The chromatin profiling in the context of aging on collected PBMCs will be performed by means of the unique CyTOF panel of the multiple (30-35) different chromatin marks. The CyTOF with the modification-specific antibodies will determine the total level of the chromatin modifications that show changes with age. Once the specific modifications are defined, the ChipSeq analysis for these modifications will be performed between young and old categories.

To further investigate the epigenetic changes with age, the Whole Genome Bisulfite Sequencing (WGBS) will be performed on purified CD14+CD16- monocytes for all donors.

Pure CD14+CD16− monocytes from young and old individuals will be profiled using deep RNA-sequencing to explore intracellular signatures.

Furthermore, the blood samples of 80 patients with Covid-19 will be profiled along with the healthy cohort using the same protocols to investigate both aging mechanisms and the influence of the COVID-19 in the diseased cohort.

Extended Pilot Project Results

Year 1 (2018/2019)

The collection of blood samples for the first year is complete. The profiling of all samples for the first year, in total for 126 subjects, has been finished. The major efforts are now focused on the analysis of these data.

Initial application of the unique cell type immunophenotyping CyTOF panel and single-cell RNA-seq profiling is a part of the manuscript that has been submitted to Immunity.

Year 2 (2019/2020)

The collection of blood samples for the second year is complete. In total, 110 samples were collected. The profiling of all samples for the second year is ongoing.

COVID-19 pandemic that emerged in 2020 demonstrated a unique age-dependent susceptibility profile. To increase public safety, Aging Biology Foundation has extended this project to include profiling of the ~70 COVID19 blood samples collected at the Washington University School of Medicine. The manuscript with a joint analysis of the cross-sectional healthy aging cohort and COVID19 patients cohort is currently in preparation.

Year 3 (2020/2021)

The collection of blood samples for the third year is in process.

The results of the Pilot Study have been published in the inaugural issue of Nature Aging. The article reports a comprehensive characterization of healthy aging in human classical monocytes, with a focus on epigenomic, transcriptomic and proteomic alterations, as well as the corresponding proteomic and metabolomic data for plasma. The samples were collected for healthy cohorts of 20 young and 20 older males (~27 and ~64 years old on average).

A new subpopulation of age-associated granzyme K (GZMK)-expressing CD8+ T-cells was identified and described in mice and humans. Human data include epigenomic, transcriptomic and proteomic signatures for 11 old vs. 11 young individuals. The manuscript has been published in Immunity by Cell Press.

Year 4 (2021/2022)

The collection of blood samples for the third year is complete. In total, 81 samples were collected.

The cellular and soluble determinants of coronavirus disease 2019 (COVID-19) relative to aging were examined by performing mass cytometry in parallel with clinical blood testing and plasma proteomic profiling. Distinct cell populations were associated with age and with COVID-19. A unique population of CD4+ T cells was associated with individuals with COVID-19 who experienced moderate, rather than severe or lethal, disease. The manuscript has been published in Nature Aging.

A comprehensive review of recent studies investigating how the immune system ages using unbiased profiling techniques at single-cell resolution has been published in Nature Reviews Immunology. The article discusses the emergent understanding of age-related alterations in innate and adaptive immune cell populations, antigen receptor repertoires, and immune cell-supporting microenvironments of the peripheral tissues.

Year 5 (2022/2023)

A profiling of the collected samples is in process.

The Artyomov Lab has contributed its expertise in immune aging and data analysis to a collaborative project investigating the effects of caloric restriction in humans. The data revealed important regulators of the immune system rejuvenation that mediate the beneficial effects of calorie restriction. The results have been published in Science.

Given the large differences in the immune system between young and old, it is an exciting and challenging task to move beyond descriptive studies of immune aging and understand the functional impact of aging on the function of immune cells, particularly T cells. A recent review summarising the most up-to-day knowledge in this area has been accepted in principle by Seminars in Immunology and published online.

Year 6 (2023/2024)

In order to complete the profiling and analysis of all collected data, a no-cost extension of the project has been agreed until the end of March 2026.

Multidimensional cytometry was performed on all PBMC samples collected (317). All samples were profiled using a dedicated 32-plex panel.  Overall, the cytometry data validate and extend the picture that emerged from the scRNA-sequencing profiling. The data are currently being further analyzed.

Whole genome DNA methylation in aging monocytes:

Data have been received for all 330 samples profiled at the WGBS level. Analysis is ongoing and figures are being compiled.

Comprehensive, large-scale single-cell profiling of healthy human blood at different ages is one of the critical pending tasks required to establish a framework for a systematic understanding of human aging. As part of this project, single-cell RNA/TCR/BCR-sequencing with protein feature barcoding was performed, and 317 blood samples from 166 healthy individuals aged 25-85 years old were profiled. In total, the dataset spanning ~2 million cells was assembled, describing 55 subpopulations of blood immune cells, with 12 subpopulations changing with age, many of which were identified and described for the first time. This work provides novel insights into healthy human aging and an annotated resource of unprecedented depth. The manuscript has been accepted by Immunity by Cell Press.