Remote immune processes revealed by immune-derived circulating cell-free DNA
Abstract
Blood cell counts often fail to report on immune processes occurring in remote tissues. Here we use immune cell type-specific methylation patterns in circulating cell-free DNA (cfDNA) for studying human immune cell dynamics. We characterized cfDNA released from specific immune cell types in healthy individuals (N=242), cross sectionally and longitudinally. Immune cfDNA levels had no individual steady state as opposed to blood cell counts, suggesting that cfDNA concentration reflects adjustment of cell survival to maintain homeostatic cell numbers. We also observed selective elevation of immune-derived cfDNA upon perturbations of immune homeostasis. Following influenza vaccination (N=92), B-cell-derived cfDNA levels increased prior to elevated B-cell counts and predicted efficacy of antibody production. Patients with Eosinophilic Esophagitis (N=21) and B-cell lymphoma (N=27) showed selective elevation of eosinophil and B-cell cfDNA respectively, which were undetectable by cell counts in blood. Immune-derived cfDNA provides a novel biomarker for monitoring immune responses to physiological and pathological processes that are not accessible using conventional methods.
Data availability
All data generated or analyzed during this study are included in the manuscript and supporting files.The whole-genome bisulfite sequencing data reported in the paper, from 46 samples, is uploaded to GEO as described. The paper also reports data from PCR reactions that were analyzed by massively parallel sequencing. This is a very large set of data that is extremely low in information content and is of little interest to readers or even to people interested in replicating our results or interrogating them further. The key information (methylation status) in each sample is provided in the supplementary information, and we also uploaded the analysis algorithm and some sequence data. The entire set of raw sequencing data is available in the Dor lab to anyone interested.Please contact Prof. Yuval Dor dor@huji.ac.il . All information will be shared. There is no need for any paperwork.Code is uploaded to GitHub as described in the paper.The methylation status of each marker in each sample is provided in Supplementary file 1. This data was used to generate the graphs shown in the paper. Sheets in this file indicate which figure they relate to.
Article and author information
Author details
Funding
No external funding was received for this work.
Reviewing Editor
- Iwijn De Vlaminck
Ethics
Human subjects: This study was conducted according to protocols approved by the Institutional Review Board at each study site (Hadassah Medical Center: HMO-14-0198. A Method to Diagnose Cell Death Based on Methylation Signature of Circulating Cell-Free DNA, Cininnati Children's Hospital: CCHMC IRB protocol 2008-0090: Eosinophils and Inflammation, an Expanded Study), with procedures performed in accordance with the Declaration of Helsinki. Blood and tissue samples were obtained from donors who have provided written informed consent. When using material from deceased organ donor those with legal authority were consented. Subject characteristics are presented in Supplementary File 1.
Version history
- Received: May 19, 2021
- Accepted: November 24, 2021
- Accepted Manuscript published: November 29, 2021 (version 1)
- Version of Record published: December 7, 2021 (version 2)
Copyright
© 2021, Fox-Fisher et al.
This article is distributed under the terms of the Creative Commons Attribution License permitting unrestricted use and redistribution provided that the original author and source are credited.
Metrics
-
- 3,579
- views
-
- 497
- downloads
-
- 28
- citations
Views, downloads and citations are aggregated across all versions of this paper published by eLife.
Download links
Downloads (link to download the article as PDF)
Open citations (links to open the citations from this article in various online reference manager services)
Cite this article (links to download the citations from this article in formats compatible with various reference manager tools)
Further reading
-
- Genetics and Genomics
- Neuroscience
Adenine phosphoribosyltransferase (APRT) and hypoxanthine-guanine phosphoribosyltransferase (HGPRT) are two structurally related enzymes involved in purine recycling in humans. Inherited mutations that suppress HGPRT activity are associated with Lesch–Nyhan disease (LND), a rare X-linked metabolic and neurological disorder in children, characterized by hyperuricemia, dystonia, and compulsive self-injury. To date, no treatment is available for these neurological defects and no animal model recapitulates all symptoms of LND patients. Here, we studied LND-related mechanisms in the fruit fly. By combining enzymatic assays and phylogenetic analysis, we confirm that no HGPRT activity is expressed in Drosophila melanogaster, making the APRT homolog (Aprt) the only purine-recycling enzyme in this organism. Whereas APRT deficiency does not trigger neurological defects in humans, we observed that Drosophila Aprt mutants show both metabolic and neurobehavioral disturbances, including increased uric acid levels, locomotor impairments, sleep alterations, seizure-like behavior, reduced lifespan, and reduction of adenosine signaling and content. Locomotor defects could be rescued by Aprt re-expression in neurons and reproduced by knocking down Aprt selectively in the protocerebral anterior medial (PAM) dopaminergic neurons, the mushroom bodies, or glia subsets. Ingestion of allopurinol rescued uric acid levels in Aprt-deficient mutants but not neurological defects, as is the case in LND patients, while feeding adenosine or N6-methyladenosine (m6A) during development fully rescued the epileptic behavior. Intriguingly, pan-neuronal expression of an LND-associated mutant form of human HGPRT (I42T), but not the wild-type enzyme, resulted in early locomotor defects and seizure in flies, similar to Aprt deficiency. Overall, our results suggest that Drosophila could be used in different ways to better understand LND and seek a cure for this dramatic disease.
-
- Genetics and Genomics
PARP-1 is central to transcriptional regulation under both normal and stress conditions, with the governing mechanisms yet to be fully understood. Our biochemical and ChIP-seq-based analyses showed that PARP-1 binds specifically to active histone marks, particularly H4K20me1. We found that H4K20me1 plays a critical role in facilitating PARP-1 binding and the regulation of PARP-1-dependent loci during both development and heat shock stress. Here, we report that the sole H4K20 mono-methylase, pr-set7, and parp-1 Drosophila mutants undergo developmental arrest. RNA-seq analysis showed an absolute correlation between PR-SET7- and PARP-1-dependent loci expression, confirming co-regulation during developmental phases. PARP-1 and PR-SET7 are both essential for activating hsp70 and other heat shock genes during heat stress, with a notable increase of H4K20me1 at their gene body. Mutating pr-set7 disrupts monomethylation of H4K20 along heat shock loci and abolish PARP-1 binding there. These data strongly suggest that H4 monomethylation is a key triggering point in PARP-1 dependent processes in chromatin.