Elevated cfDNA after exercise is derived primarily from mature neutrophils, with a minor contribution of cardiomyocytes, 2023, Fridlich et al

SNT Gatchaman · Jul 30, 2023

Elevated cfDNA after exercise is derived primarily from mature polymorphonuclear neutrophils, with a minor contribution of cardiomyocytes
Ori Fridlich; Ayelet Peretz; Ilana Fox-Fisher; Sheina Pyanzin; Ziv Dadon; Eilon Shcolnik; Ronen Sadeh; Gavriel Fialkoff; Israa Sharkia; Joshua Moss; Ludovica Arpinati; Shachar Nice; Christopher D. Nogiec; Samuel Terkper Ahuno; Rui Li; Eddie Taborda; Sonia Dunkelbarger; Zvi G. Fridlender; Paz Polak; Tommy Kaplan; Nir Friedman; Benjamin Glaser; Ruth Shemer; Naama Constantini; Yuval Dor

Strenuous physical exercise causes a massive elevation in the concentration of circulating cell-free DNA (cfDNA), which correlates with effort intensity and duration. The cellular sources and physiological drivers of this phenomenon are unknown.

Using methylation patterns of cfDNA and associated histones, we show that cfDNA in exercise originates mostly in extramedullary polymorphonuclear neutrophils. Strikingly, cardiomyocyte cfDNA concentration increases after a marathon, consistent with elevated troponin levels and indicating low-level, delayed cardiac cell death. Physical impact, low oxygen levels, and elevated core body temperature contribute to neutrophil cfDNA release, while muscle contraction, increased heart rate, b-adrenergic signaling, or steroid treatment fail to cause elevation of cfDNA. Physical training reduces neutrophil cfDNA release after a standard exercise, revealing an inverse relationship between exercise-induced cfDNA release and training level.

We speculate that the release of cfDNA from neutrophils in exercise relates to the activation of neutrophils in the context of exercise-induced muscle damage.

Highlights

The major source of elevated cfDNA in exercise is mature polymorphonuclear neutrophils

Physical impact and elevated core body temperature contribute to cfDNA elevation

cfDNA elevation has an inverse relationship to individual training level

A minor contribution of cardiomyocyte-derived cfDNA after extreme exercise

Link | PDF (Cell Reports Medicine)

SNT Gatchaman · Jul 30, 2023

See also the preceding Physical activity specifically evokes release of cell-free DNA from granulocytes thereby affecting liquid biopsy (2022, Clinical Epigenetics)

RedFox · Jul 30, 2023

Dying heart cells? That sounds scary. But strenuous exercise is good for you. Do they just grow back?

Andy · Jul 30, 2023

Interesting. I searched for studies looking for cell-free DNA in ME/CFS patients and found one that found no difference with controls, Analysis of 16S rRNA gene sequences and circulating cell-free DNA from plasma of chronic fatigue syndrome and non-fatigued subjects 2002 Vernon et al,

and one that found elevated rates in patients, and one that found elevated rates, Mitochondrial dysfunction and the pathophysiology of Myalgic Encephalomyelitis/Chronic Fatigue Syndrome (ME/CFS) 2012 Booth et al

Hutan · Jul 31, 2023

Circulating cell-free DNA (cfDNA) fragments are released from dying cells to blood. While the exact mechanisms of release are poorly understood, it is clear that cfDNA fragments are typically nucleosome size (167 bp on average), and that their half-life in circulation is 15–60 min.6,7 Healthy individuals have ∼1,000 genome equivalents per milliliter of plasma (GE/mL), originating mostly in blood cells.8 The concentration of cfDNA is elevated in multiple pathologies such as trauma, infection, myocardial infarction and cancer, reflecting excessive cell death in the immune system and in affected solid tissues.9,10,11
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That's a short half-life

The reported increase can be massive (up to 20-fold)16 but persists only for the duration of exercise, with a rapid decline upon cessation of activity.17
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Perhaps it would be necessary to look for differences in cell-free DNA immediately after exercise and/or during PEM.

Hutan · Jul 31, 2023

We and others have established a technology to determine the tissue origins of cfDNA, based on the detection of cell-type-specific DNA methylation patterns in plasma. Using an extensive atlas of human cell type methylomes, we were able to identify genomic loci that are methylated or unmethylated uniquely in specific cell types; analysis of the methylation patterns of such loci in cfDNA provides a highly specific and sensitive indicator for the tissue origins of cfDNA in health and disease.21,22,23,24,25More recently, we developed a method to study the patterns of histone modifications on circulating chromatin, using cell-free chromatin immunoprecipitation (cfChIP) followed by sequencing (cfChIP-seq). Capturing circulating chromatin fragments that associate with histone marks of gene activity allows us to infer gene expression programs in the cells that released cfDNA.26
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So, a new technique to identify the source of cell free DNA. There might be issues if the cells of people with ME/CFS that then go on to release cell free DNA are not operating normally (and so are methylated unusually), but it still sounds interesting.

Hutan · Jul 31, 2023

A graded exercise test caused on average a 5.5-fold increase in cfDNA concentration, while a 40-min run at anaerobic threshold, a half marathon, and a full marathon led to a respective 18-, 23-, and 39-fold increase on average compared with baseline (Figure 1A). The elevation was short lived: 1 h after exercise, cfDNA levels returned to near baseline (Figure 1B), consistent with a previous study.
13
The extent of cfDNA elevation after a CPET or a 40-min run varied considerably between individuals (5- to 40-fold relative to baseline), even though the experiments were designed to demand a similar aerobic effort, suggesting that parameters other than intensity underlie cfDNA release in exercise.
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Those are massive increases in levels of cfDNA. The graded exercise test is just 12 minutes, a CPET. It's interesting that there was a lot of variation in fold change among the participants.

These figures (1C and 1D) show the amount of cfDNA released by various cell types in a range of resting/exercise states. Sample sizes are low. I would really like to see this approach applied to people with ME/CFS. Among the cell type sources that are claimed to be assessed are vascular endothelial cells and colon cells. The cfDNA derived from neutrophils is shown in red. If this study is right, it really is remarkable just how much DNA is coming from the neutrophils in response to exercise (and they seem to be suggesting it's specifically a response to hypoxic conditions).

What could that mean for a person with ME/CFS? Could PEM be a response to the loss of neutrophils? Or to the low oxygen levels?

Is the sickness response that happens when someone has a fever a similar thing?

How could a massive turnover of neutrophils be a good thing to happen in response to an exercise challenge?

Hutan · Jul 31, 2023

On the finding that physical impact seems to increase cf-DNA (whereas an increased heart rate does not)

To further isolate the impact of muscle activity, we obtained plasma from subjects performing EMS training. In EMS, muscle activity is elicited by external electrical stimulation, with minimal cardiovascular involvement.
55
As shown in Figure 5E, a 25-min session of EMS (n = 11 subjects) did not elevate cfDNA levels at all, indicating that muscle activity is not sufficient to drive cfDNA elevation in exercise.

Physical impact
When comparing different types of physical activity, we observed that running led to a higher level of cfDNA than swimming and cycling, even when the latter two were performed at extreme intensity. As shown in Figure 6A, a half marathon led on average to 55-fold increase in neutrophil cfDNA, while a 10-km open-water swim (European championship) led on average to a 24-fold increase, and a 32-km bicycle time-trial race (national championship) elevated neutrophil cfDNA only 10-fold.
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Could this, together with the finding that an increased body temperature increases cf-DNA, be why some people with ME/CFS prefer swimming over other exercising - less impact and less over-heating?

However, untrained individuals (>4 mM lactate or >70%HRMax) had a significantly higher elevation of neutrophil cfDNA compared with trained individuals (Figure 7B), supporting the idea that aerobic training attenuates cfDNA release in exercise.
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They found lower levels of neutrophil cfDNA in trained individuals compared to untrained individuals.

SNT Gatchaman · Jul 31, 2023

Hutan said: ↑

They found lower levels of neutrophil cfDNA in trained individuals compared to untrained individuals.
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Which I think relates to DNase activity. From Physical Exercise Promotes DNase Activity Enhancing the Capacity to Degrade Neutrophil Extracellular Traps (2022, Biomedicines) —

Importantly, not only the release of cfDNA but also its elimination from the circulation determines plasma concentrations. Higher DNase activity is regarded as a beneﬁcial mediator reducing the pro-inﬂammatory and pro-thrombotic potential of cfDNA and NETs by timely degradation. As major waste-management endonucleases, different DNases exist to catalyze hydrolysis of DNA in circulation or intracellularly, e.g., during apoptosis.
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Insufﬁcient degradation of cfDNA due to inadequate activity of DNase1 provokes hemostatic turbulences and tissue damage, as well as thrombosis or immune complex formation.
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At ﬁrst, we speculated that increased DNase activity might result from the body’s effort to counter regulate exercise-provoked release of cfDNA and sub-clinical chronic inﬂammation [...] However, our data show a disproportionally higher increase in DNase activity, as evidenced by the cfDNA/DNase ratio, mirroring an adjusted balance in response to consequent exercise with actual performance gain.
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This observation could suggest that physical activity reduces the relative exposure to cfDNA by an increase in total DNase activity, limiting the immunogenic and pro-inﬂammatory risks. Importantly, neither absolute levels of cfDNA and DNase nor the relative change correlated, indicating once more an uncoupled constant increase in DNase activity, not exclusively in response to acute DNA release.
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SNT Gatchaman · Jul 31, 2023

That paper also comments —

DNase1 can be produced by several tissues, foremost by liver and intestines, while DNase1L3 is predominantly released by cells of the myeloid lineage. Recently, it has been shown that increased total cholesterol levels above 200 mg/dL attenuate a timely DNase response to pro-inﬂammatory stimuli by inducing endoplasmic reticulum (ER)-stress..
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So lipid dysregulation may impair control of cfDNA / NETs via DNases.

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Elevated cfDNA after exercise is derived primarily from mature neutrophils, with a minor contribution of cardiomyocytes, 2023, Fridlich et al

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