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Hummel et al. Translational Psychiatry (2018) 8:236
DOI 10.1038/s41398-018-0264-x
T
ranslational Psychiatry
ARTICLE Open Access
Cell-free DNA release under psychosocial
and physical stress conditions
E. M. Hummel
1
,E.Hessas
1
,S.Müller
1
,T.Beiter
2
,M.Fisch
3
,A.Eibl
3
,O.T.Wolf
4
,B.Giebel
5
,P.Platen
3
,R.Kumsta
1
and
D. A. Moser
1
Abstract
The understanding of mechanisms linking psychological stress to disease risk depend on reliable stress biomarkers.
Circulating cell-free DNA (cfDNA) has emerged as a potential biomarker of cellular stress, aging, inflammatory
processes, and cell death. Recent studies indicated that psychosocial stress and physical exercise might also influence
its release. We compared the effects of acute psychosocial and physical exercise stress on cfDNA release by exposing
20 young, healthy men to both an acute psychosocial laboratory stressor and an acute physical exercise stressor.
Venous blood and saliva samples were collected before and after stress exposure. Cell-free DNA was extracted from
plasma and quantified by qPCR. Furthermore, cfDNA fragment length was analyzed and cfDNA methylation patterns
were assayed across time. In addition, release of stress hormones and subjective stress responses were measured.
Results showed a twofold increase of cfDNA after TSST and fivefold increase after exhaustive treadmill exercise, with an
overabundance of shorter cfDNA fragments after physical exhaustion. Interestingly, cell-free mitochondrial DNA
showed similar increase after both stress paradigms. Furthermore, cfDNA methylation signatures—used here as a
marker for diverse cellular origin—were significantly different post stress tests. While DNA methylation decreased
immediately after psychosocial stress, it increased after physical stress, suggesting different cellular sources of active
DNA release. In summary, our results suggest stimulus and cell-specific regulation of cfDNA release. Whereas the
functional role of stress-associated cfDNA release remains elusive, it might serve as a valuable biomarker in molecular
stress research as a part of the psychophysiological stress response.
Introduction
Increased levels of circulating cell-free DNA (cfDNA) in
the bloodstream, either of genomic or mitochondrial
origin, are hallmark manifestations of acute systemic
inflammatory responses as well as of chronic inflamma-
tion. Elevated levels have been reported after trauma,
sepsis, stroke, ischemia/reperfusion injury, or myocardial
infarction, and in patients suffering from cancer, auto-
immune and cardiovascular diseases, as well as metabolic
disorders
1–4
. In these conditions, cfDNA has by now been
established as a reliable and reproducible biomarker, and
quantification of cfDNA levels offers potential as a pro-
mising clinical analyte in risk profiling and therapy
monitoring in diverse inflammatory settings. Apart from
pathological conditions, it has recently been observed that
physical exercise acutely triggers an immediate and
transient increase in cfDNA
5–9
but also that exposure to
chronic psychosocial stress influences plasma cfDNA
levels
10–12
. Both acute bouts of exercise and acute psy-
chosocial stressors provoke immediate neuroendocrine,
inflammatory, metabolic, and cardiovascular responses
that impact immune homeostasis at multiple levels. It
thus appears conceivable that psychosocial stress and
mental health conditions should also acutely or chroni-
cally impact cfDNA plasma levels. Indeed, recent studies
indicate that cfDNA could be a valuable biomarker in the
© The Author(s) 2018
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Correspondence: D. A. Moser (dirk.moser@rub.de)
1
Department of Genetic Psychology, Faculty of Psychology, Ruhr-University
Bochum, Universitätsstraße 150, 44801 Bochum, Germany
2
Department of Sports Medicine, Medical Clinic, Eberhard-Karls-University of
Tübingen, Otfried-Müller-Straße 10, 72076 Tübingen, Germany
Full list of author information is available at the end of the article.
These authors contributed equally: E.Hummel, E. Hessas
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context of psychosocial stress and dysfunction. Increased
plasma cfDNA levels were found in animals after expo-
sure to emotional stress as provoked by tail fixation for
18 h
13
. Women undergoing in vitro fertilization treatment
who reported high levels of psychosocial distress have
been reported to show elevated levels of cfDNA which
could be lowered by means of stress reduction interven-
tion
10–12
. Interestingly, recent research has also identified
increased numbers of mitochondria following stressful
events and during depression, an effect that is thought to
be mediated by stress hormones activated through the
hypothalamic–pituitary–adrenal (HPA) axis
14,15
. Altered
levels of circulating cell-free mitochondrial DNA (cf-
mtDNA) in the plasma of suicide attempters and in major
depressive disorder have also been described
16–18
.In
addition, increased plasma cf-mtDNA levels in suicide
attempters were significantly and positively correlated
with cortisol levels after dexamethasone suppression, an
indicator of hyperactivity of the HPA axis, the organism’s
major hormonal stress response system
17
.
To date, cfDNA origin, mechanisms of release, regula-
tion, clearance, and its physiological role are still unclear.
Fragment sizes ranging from ~150 bp to larger than
10 kbp have been observed
19,20
. Fragments of 150 bp and
multiples of 150 bp are thought to derive from apoptotic
processes originating from the endogenous cleavage of
chromatin DNA into inter-nucleosomal fragments
21
,
whereas larger fragments of ≥10 kbp are thought to derive
from necrotic processes. However, active DNA release
during disease and after stimulation has also been
observed. For instance, cfDNA was found in cell culture
supernatant and might act as a potential signaling mole-
cule under distinct conditions
19,22
. Another source of
cfDNA are neutrophil extracellular traps (NETs) which
represent an ancient and important part of our innate
immune defense system
23,24
. NETs are composed of
remodeled extracellular DNA fibers that are released by
neutrophils in response to pathogenic triggers. Moreover,
several white blood cell lineages have by now been
reported to be capable of actively releasing DNA, either
from nuclear or mitochondrial genomic material
25,26
.
Given the associations between increased cfDNA levels
and chronic stress as well as markers of stress system
dysregulation, we aimed to test whether and to what
extent acute psychosocial and physical stress exposure
might lead to increased levels of cfDNA and cf-mtDNA in
the circulation. We also aimed to test for potential
stimulus-specific effects and compared lengths of corre-
sponding cfDNA fragments and their specific methylation
pattern as an indicator of different cellular origin after
acute psychosocial to physical exercise stress. Lastly, we
associated cfDNA levels with release patterns of HPA axis
and sympathetic nervous system markers to identify
potential release triggers. In addition, emotional responses
were measured by means of self-report questionnaires.
Materials and methods
Participants
Participants (n=20) were healthy male students of
sports science, 18 to 36 years of age (mean =23.3 ± 3.8
(SD)), with a normal body mass index (mean =23.4 ± 1.5),
no history of or current mental health problems as well as
no chronic or acute physical illnesses, and no current
intake of medication. All participants gave written
informed consent and the study was approved by the local
ethics committee (153/2014).
Procedure
Participants were exposed to both an acute psychosocial
laboratory stressor and an acute physical exercise stressor
in a randomized order on two different days. Sessions
were scheduled for either 9 or 11 am to keep variations in
the diurnal cycle of cortisol at a minimum. Induction of
psychosocial and exercise stress and the accompanying
testing sessions were carried out at least 2 days apart. Half
of the participants completed the Trier social stress test
(TSST) first, while the other half went through the exer-
cise protocol first, and the order of testing was assigned
pseudo-randomly. On arrival, participants filled out a self-
report questionnaire on their health status in regard to
exercise (Physical Activity Readiness Questionnaire (PAR-
Q)
27
) which was reviewed by one of the sports medical
physicians on site. A peripheral venous catheter was then
inserted on the inside of the participant’s elbow or on
their hand by a medical physician (45 min before stress
induction), after which participants filled out ques-
tionnaires for approximately 25 min, followed by a resting
period until the respective stress protocol started. Blood
and saliva were sampled 2 min before and 2, 15, 30, and
40 min after cessation of the respective protocol. At four
time points (−2, +2, +15, and +30 min), the Social
Emotional Response Scale (SERS; Schlotz and Kumsta,
unpublished) was completed by the participants. The
questionnaire includes 15 questions for the evaluation of
arousal (calm, jittery, tense, intense, relaxed, content),
self-directed emotions (guilty, ashamed, blameworthy,
angry at self, dissatisfied with self), and anxiety (fearful,
worried), rated on a scale ranging from 1 =not at all to
4=a lot.
Induction of psychosocial stress
Psychosocial stress was induced by means of the TSST
as described elsewhere
28
. In brief, the TSST consists of a
preparation period, a free speech, and an unanticipated
math task performed in front of a panel of judges and a
camera. The TSST is a very well-validated and widely
Hummel et al. Translational Psychiatry (2018) 8:236 Page 2 of 10
used standardized acute laboratory stressor. As such, it
has repeatedly been shown that it reliably activates the
HPA axis and in turn leads to significant elevations of the
stress hormone cortisol
29
which has been attributed in
large part to the elements of uncontrollability and social-
evaluative threat immanent to the situation
30
.
Induction of physical exercise stress
An exhaustive treadmill exercise with a 15% incline was
carried out in order to keep the duration to 10 to 15 min,
as similar as possible to that of the TSST. It started out
with a 5 min walking period at 1 m/s, after which a
stepwise increase of speed by 0.2 m/s was introduced
every 30 s until the participant reached subjective
exhaustion, at which point the treadmill was stopped and
the stress induction was thus concluded.
Plasma preparation and cfDNA extraction
In a recent overview, El Messaoudi et al.
31
compared
pre-analytical factors influencing cfDNA quality from the
moment of blood drawing to storage of extracted cfDNA.
According to their recommendations, 5 ml of whole blood
was collected in EDTA-collection tubes (EDTA Monov-
ettes, Sarstedt, Germany) at each time point and was
immediately centrifuged at 1600 × gand 4 °C for 10 min.
Plasma was transferred to a fresh tube followed by a
second 10 min of centrifugation at 16,000 × gand 4 °C.
Finally, plasma was passed through a 0.8 µm filter and
aliquots were stored at −80 °C until further analysis.
The QIAamp Circulating Nucleic Acid Kit (Qiagen,
Hilden, Germany), which is considered the gold standard
for cfDNA extraction
32,33
, was used to extract cfDNA
from 0.9 ml plasma according to the manual provided
with the kit. Cell-free DNA was eluted in a final volume of
100 µl H
2
O.
Spike-in preparation
To control for constant extraction efficiencies, all
plasma samples were spiked with plasmid DNA at defined
copy numbers as described below. This spike-in control
DNA was generated from a 3493 bp pMK-RQ cloning
vector, carrying a DNA fragment of the pigeon (Columba
livia), activity regulated cytoskeleton associated protein
(Arc; XM_005510918.1; kindly provided by Dr. Rena
Klose). Plasmid copy numbers were calculated using the
DNA copy number and dilution calculator (www.
thermofisher.com). Subsequently, 2 µg plasmid DNA
was digested using Tsp45I (NEB, Frankfurt am Main,
Germany), generating fragments of 103 bp, 306 bp,
663 bp, and 2,421 bp from the circular plasmid. Reaction
was heat-inactivated at 65 °C for 15 min, diluted to 1 × 10
6
copies/µl, aliquotted, and frozen at −20 °C. Completeness
of digestion was controlled using an Agilent DNA 1000
kit on an Agilent 2100 Bioanalyzer. Each plasma sample
was spiked with 400,000 copies of fragmented plasmid
and eluted in 100 µl H
2
O, resulting in 4000 plasmid
copies/µl. Percentage efficiency of extraction was con-
trolled by quantitative polymerase chain reaction (qPCR)
targeting plasmid fragments compared to its specific
standard curves, ranging from 2.5 × 10
2
to 2.5 × 10
5
copies.
Quantitative PCR
The qPCRs for the assessment of cfDNA, cf-mtDNA,
and spike-in controls were carried out on a CFX384
Touch™Real-Time PCR Detection System (BioRad, Her-
cules, USA). Primers for cfDNA, cf-mtDNA, and spike-in
control were designed to produce target-specific ampli-
cons of 70–110 bp (see supplementary information 1).
The limits of detection (LOD) and the limits of quantifi-
cation (LOQ) were determined as described elsewhere
34
.
As pre-tests revealed that cfDNA extraction was highly
efficient for different fragment sizes (supplementary
information 2), and in order to save cfDNA material for
other applications, only the 103 bp ARC fragments were
used as a reporter of cfDNA extraction efficiency. CfDNA
displays a reasonably good representation of the whole
genome with a relative amount of all genomic features in
cfDNA of approximately 1
35
. Therefore, we used an in-
house BDNF assay (brain-derived neurotrophic factor)
that is usually used for high resolution melting (HRM)
genotyping of rs6265A/G. This in-house qPCR assay
combines high affinity with high linearity, highly satisfying
LOD/LOQ and no primers detectable by melting curve
analysis for the no-template controls (NTCs). The qPCR
reaction for the quantification of cfDNA, cf-mtDNA, and
spike-in control contained 0.2 µM primers (see supple-
mentary information 1) and 5 µl iTaq Universal SYBR
Green Supermix (BioRad, Hercules, USA) in a total
volume of 10 µl. The standard amplification protocol
included an initial denaturation step for 3 min at 95 °C,
followed by 40 cycles of melting at 95 °C for 5 s, annealing
and extension at 60 °C for 20 s, and a melting curve
analysis. Triplicate qPCR assays were performed for each
point of a cfDNA- and cf-mtDNA-specific standard curve
(generated from artificial human DNA and mtDNA gene
fragments; MWG Eurofins, Ebersberg) compared to the
unknown study samples. All experiments included NTCs
with the addition of diluent without cfDNA extract or Arc
plasmid, and plate normalization was achieved using an
interplate calibrator.
Cell-free DNA fragment analysis
To test for potential differences in cfDNA fragment
length after psychosocial vs. physical stress tests, cfDNA
samples were analyzed on a Fragment Analyzer
(Advanced Analytical, Heidelberg, Germany) using the
DNF-488 HS Genomic DNA Kit (Prerun: 6.0 kV, 30 s;
Hummel et al. Translational Psychiatry (2018) 8:236 Page 3 of 10
Sample injection: 9 kV, 30 s; Separation: 6.0 kV, 50 min).
This test enables the analysis of minute amounts of DNA
(50 pg/µl) for their respective size in the range of 50 bp to
40 kbp. Samples were analyzed in the Genomics and
Transcriptomics Laboratory of the University of Düssel-
dorf (BMFZ/GTL Professor Karl Köhrer).
DNA methylation analysis
In order to test for potential differences in cellular
origin of circulating cfDNA after stimulation, DNA
methylation patterns of the homeobox A5 gene (HOXA5)
over time were compared between conditions. HOXA5
was chosen for methylation analysis because of its highly
cell type-specific DNA methylation patterns, showing
hypermethylation in muscle cells, hypomethylation in
brain cells deriving from the hippocampus and cultured
neuronal cell lines, and differential methylation in blood
cells (http://www.roadmapepigenomics.org/;http://www.
blueprint-epigenome.eu/). cfDNA (20 µl) was subjected to
bisulfite conversion (EZ-96 DNA Methylation-Lightning™
Kit, Zymo Research) and eluted in 10 µl H
2
O. Using pri-
mers as indicated in supplementary information 1, a
173 bp HOXA5 gene fragment located in the CpG island
of exon1 (chr7:27,182,652–27,182,824) was amplified on a
T100™Thermal Cycler (BioRad, Hercules, USA). For
amplification, 2.5 µl bisulfite-modified DNA, 0.2 µM pri-
mers (see supplementary information 1), and 15 µl
GoTaq®G2 Hot Start Green Master Mix (Promega
GmbH, Mannheim, Germany) were mixed in a total
volume of 30 µl. The amplification included an initial
denaturation step of 2 min at 95 °C, followed by 50 cycles
of melting at 95 °C for 30 s, annealing at 56 °C for 45 s,
extension at 74 °C for 45 s, and a final extension step at
74 °C for 10 min. PCR products were purified using 1.5 µl
of a sepharose beads suspension (GE Healthcare, UK).
Quantitative methylation analysis was performed on a
Pyromark Q24 Advanced system (Qiagen, Hilden, Ger-
many) comparing individual DNA signatures over time
for the different stress paradigms tested. To ensure that
the assay was not biased toward methylated or non-
methylated DNA, it was validated using a standard curve
of DNA with known methylation (100%; 75%; 50%; 25%;
0%). Methylation standards were created using DNA
methyltransferase (M.SssI; NEB, Frankfurt am Main,
Germany), and non-methylated standards were generated
using the REPLI-g Mini Kit (Qiagen, Hilden, Germany)
according to the manuals.
Hormonal analysis
Plasma and salivary cortisol were analyzed on a
Synergy2 plate reader (Biotek, USA) using commercial
enzyme-linked immunosorbent assays (ELISAs; cortisol
and free cortisol in saliva; Demeditec, Germany)
according to the manufacturer’s instructions. Intra- and
interassay variability were less than 7% and 8.5% for
plasma cortisol and less than 5% and 7% for salivary
cortisol, respectively. Salivary alpha-amylase (sAA) activ-
ity
35
was measured as described elsewhere
36
, and showed
an intra- and interassay variability of less than 4% and 5%,
respectively. Catecholamines, in this case adrenaline (A)
and noradrenaline (NA), were commercially high-
performance liquid chromatography (HPLC)-measured
by LSM (Labor für Stressmonitoring, Göttingen, Ger-
many). To estimate plasma catecholamine concentrations,
a solvent extraction system for the selective and quanti-
tative isolation of A and NA from a sample matrix was
used. The clean-up procedure was adopted from Smedes
et al.
37
and slightly modified for the use of lower sample
volumes.
Statistical analysis
Statistical analysis was performed with the statistical
program SPSS (version 20, SPSS Statistics/IBM Corp.,
Chicago IL, USA). The data were analyzed with repeated
measures analysis of variance (ANOVA; with
Greenhouse–Geisser correction for violation of the
assumption of sphericity). The effect sizes were reported
as eta (ƞ2). In case of significant effects, Fisher's least
significant difference (LSD) test was carried out to check
for group differences. Some of the parameters were not
normally distributed and therefore transformed. A natural
logarithm (ln) transformation was applied to cfDNA, cf-
mtDNA, alpha-amylase, adrenaline, noradrenaline,
plasma, and salivary cortisol data.
Results
cfDNA
Quantification of circulating cfDNA revealed
significantly increased levels after both stress paradigms
(main effect time: F
(3.22, 122.49)
=101.44, p< 0.001,
η
2
=0.727). Immediately after cessation of psychosocial
stress, cfDNA significantly increased from ~8900 copies
of cfDNA per milliliter plasma (copies/ml) to more than
16,200 copies/ml. Following the physical stress condition,
afivefold increase in circulating cfDNA with peak levels at
15 min after maximal physical strain was observed (see
Fig. 1a). Magnitude of cfDNA release and response
dynamics, i.e., differences in the timing of peak levels,
differed significantly between conditions (main effect
condition: F
(1, 38)
=51.98, p< 0.001, η
2
=0.578; time ×
condition interaction effect: F
(3.22, 22.49)
=30.42, p< 0.001,
η
2
=0.445). Post-hoc test showed significant difference
between conditions at all measured time points after the
stress tests (p< 0.001). CfDNA rapidly decreased after
reaching peak levels and returned close to baseline levels
Hummel et al. Translational Psychiatry (2018) 8:236 Page 4 of 10
within the time frame tested, which is consistent with the
cfDNA half-life of ~15 min
38
.
Fragment analysis
Before stress exposure, DNA fragment analysis showed
that 6.6% of cfDNA fragments consisted of fragments
smaller than 300 bp, most of about 170 bp in size. Thus,
92.6% of the cfDNA fragments had a size between 300 bp
and 1.500 bp, and less than 1% were longer than 1500 bp,
with a maximum fragment length of 40 kbp, and were not
further analyzed (see supplementary information 3 for
fragment abundance over time). As shown in Fig. 1b, the
~170 bp fragments increased significantly after both stress
conditions. Repeated measures ANOVA showed a sig-
nificant effect of time (F
(3.04, 115.65)
=36.45, p≤0.001, η
2
=0.490), an effect of condition (F
(1, 38)
=35.84, p≤0.001,
η
2
=0.485), and a time by condition interaction (F
(3.04,
115.65)
=33.77, p≤0.001, η
2
=0.471) for the ~170 bp
fragments. Post-hoc test showed significant difference
between conditions at time points +15 min, +30 min, and
0
50
54
57
60
63
66
Methylation HOXA5
psychosocial vs. physical stress
Time (min)
Methylation HOXA5 (%)
physical stress
psychosocial stress
(C)
STRESS
TEST
*
0
cf-mtDNA
psychosocial vs. physical stress
Time (min)
cf-mtDNA (copies/ml)
psychosocial stress
physical stress
(D)
STRESS
TEST
*
0
6
×
104
5
×
104
1.5
×
106
1.2
×
106
9
×
105
6
×
105
3
×
105
4
×
104
3
×
104
2
×
104
1
×
104
cfDNA
psychosocial vs. physical stress
-2 +2 +15 +30 +40 -2 +2 +15 +30 +40
-2 +2 +15 +30 +40 -2 +2 +15 +30 +40
Time (min)
cfDNA (copies/ml)
psychosocial stress
physical stress
(A)
STRESS
TEST
***
***
***
***
0.0
2.5
5.0
7.5
10.0
12.5
15.0
170 bp Fragments
psychosocial vs. physical stress
Time (min)
170 bp Fragments (%)
psychosocial stress
physical stress
(B)
STRESS
TEST
***
***
***
Fig. 1 cfDNA (concentration, methylation, fragment length) and cf-mtDNA concentrations before and after psychosocial and physical
stress are shown. a Changes in cfDNA concentrations (copies/ml plasma) before and after psychosocial and physical stress. Immediately after the
TSST, doubling of cfDNA concentration was observed, whereas it increased fivefold after physical stress, peaking 15 min after the cessation of
exercise. bChanges in the percentage proportion of the 170 bp cfDNA fragments of purified cfDNA. While minimal increases of the 170 bp fragments
could be observed after the TSST, a fivefold percentage increase 15 min after physical exercise was observed. cPercentage cfDNA methylation of a
HOXA5 fragment is shown as the average of 9 CpG sites. DNA methylation increased by 7.5% after physical stress, while it significantly decreased by
6.5% after the TSST. dAn almost twofold increase of cf-mtDNA directly after both stress conditions is shown. Post-hoc test showed significant
difference between the stress paradigms at time point +15 min. Values are reported as means ± SEM. The data were analyzed with repeated
measures ANOVAs. In case of significant effects, post-hoc test was carried out to check for group differences (*p≤0.05, ***p≤0.001)
Hummel et al. Translational Psychiatry (2018) 8:236 Page 5 of 10
+40 min after stress test (p< 0.001). Analysis for the
fragments between 300 and 1500 bp showed no significant
effects.
Epigenetic analysis of HOXA5-specific cfDNA fragments
In order to test for differences in cellular origin of
cfDNA between conditions, we used HOXA5 as a reporter
gene, as it displays significant tissue-specific DNA
methylation. As shown in Fig. 1c, changes in DNA
methylation patterns over time followed opposite
directions (time × condition interaction effect: F
(4, 120)
=
7.63, p< 0.001, η
2
=0.203; effect of time: F
(4, 120)
=1.68,
p=0.159, η
2
=0.053; effect of condition: F
(1, 30)
=0.006,
p=0.94, η
2
=0.000). Post-hoc test showed significant
difference between conditions directly after the stress
tests (p=0.033). After TSST, DNA methylation
decreased from baseline by 6.5% before it increased again,
whereas it increased by 7.5% after physical exercise.
Cf-mtDNA
Immediately after both stress paradigms, significant
elevations of cf-mtDNA were observed (effect of time:
F
(3.14, 119.18)
=4.82, p=0.003, η
2
=0.113; time × condition
interaction effect: F
(3.14, 119.18)
=1.60, p=0.192, η
2
=
0.040; effect of condition: F
(1, 38)
=1.80, p=0.188, η
2
=
0.045), with a 1.7-fold increase after TSST and 1.6-fold
increase after physical stress. After both stress situations
cf-mtDNA decreased rapidly back to baseline levels
within 30 min, as shown in Fig. 1d. A significant difference
between conditions at time point +15 min was found in
the post-hoc test (p=0.035).
Hormonal activation
Significant increases were observed for all hormones
(effect of time: all F> 13.19, all p< 0.001, η
2
> 0.258; see
Fig. 2for response curves). Furthermore, we observed
differences in magnitude and reaction curve patterns
between conditions for all measures, with larger increases
following physical exercise (time × condition interaction
effect: all F> 11.10, all p< 0.001, all η
2
> 0.226; main effect
condition: all F> 2.20, all p< 0.146, all η
2
> 0.055), except
for sAA (see supplementary information 4).
Emotional response
Analysis of psychosocial responses showed an increase
in tense arousal following both stress situations (effect of
time: F
(2.20, 81.34)
=66.85, p< 0.001, η
2
=0.644; effect of
condition: F
(1, 37) =
5.81, p=0.021, η
2
=0.136; time ×
condition interaction effect: F
(2.20, 81.34)
=2.72, p=0.067,
η
2
=0.069). Post-hoc test showed significant difference
between conditions at time points +2 min (p=0.013) and
+15 min (p=0.008). Self-directed emotions and anxiety,
however, increased only during psychosocial stress con-
ditions, whereas these emotions remained unchanged
after physical stress (self-directed emotions: effect of time:
F
(1.84, 67.95)
=13.38, p< 0.001, η
2
=0.266; effect of
condition: F
(1, 37)
=6.75, p=0.013, η
2
=0.154; time ×
condition interaction effect: F
(1.84, 67.95)
=6.76, p=0.003,
η
2
=0.154; anxiety: effect of time: F
(2.10, 77.70)
=4.11, p=
0.019, η
2
=0.100; effect of condition: F
(1, 37)
=9.21 p=
0.004, η
2
=0.199; time × condition interaction effect:
F
(2.10, 77.70)
=2.87, p=0.060, η
2
=0.072; see supplemen-
tary information 5). Post-hoc test showed significant dif-
ferences between conditions for self-directed emotions
directly after stress condition (p< 0.001) and for anxiety at
time points −2 min (p=0.004), +2 min (p=0.003), and
+15 min (p=0.018).
Correlation between hormones and cfDNA
As illustrated in Fig. 3a, a significant positive correlation
was observed between salivary cortisol increase and cfDNA
increase in the physical stress condition (r=0.539, R
2
=
0.291, p=0.014), with a similar relationship between
plasma cortisol increase and cfDNA increase at trend level
(Fig. 3b: r=0.419, R
2
=0.176, p=0.066). Correlations with
indicators of SNS activity and cfDNA did not
show significant effects, although there was a positive
association between cfDNA increase and adrenaline
increase in the physical stress condition (r=0.381, R
2
=
0.145, p=0.097). There were no significant correlations
between any of the hormones and cfDNA in the TSST
condition.
Correlation between hormones and cf-mtDNA
No correlation was observed between increases in cf-
mtDNA and cortisol or catecholamines in either condi-
tion. However, when considering individual measuring
points, a correlation between cf-mtDNA and adrenaline
15 min after physical stress could be found (r=0.451,
R
2
=0.203, p=0.046).
Discussion
Stress-related mental and physical disorders have seen a
continuous rise in recent years and entail a tremendous
societal socio-economic burden. The understanding of the
mechanisms linking psychosocial stress to disease risk
depend on reliable stress biomarkers. Increased levels of
cell-free DNA, an emerging biomarker for a range of
pathological conditions, have recently been associated with
the experience of chronic stress. Here, we show for the
first time that exposure to acute psychosocial stress leads
to immediate and transient increases in cfDNA levels.
In contrast to cfDNA, the magnitude of cf-mtDNA
release was similar between psychosocial and physical
provocation. Mitochondria express glucocorticoid recep-
tors
39
providing for a potential functional link between
stress exposure and cf-mtDNA biology. Recently, elevated
cf-mtDNA levels in plasma were found in individuals who
Hummel et al. Translational Psychiatry (2018) 8:236 Page 6 of 10
had attempted suicide, and these blood cf-mtDNA levels
correlated with high post-dexamethasone cortisol levels
16
,
supporting an association between HPA axis activity and
mitochondrial function. In our study of healthy young men,
however, no association could be observed between cortisol
and cf-mtDNA release in either condition. Cf-mtDNA
levels have also been assessed in patients diagnosed with
mental disorders, with mixed results. In patients with major
depressive disorder, both elevated
17,18
and decreased
levels
16
have been reported. Furthermore, Jiang et al.
40
recently reported increased mtDNA levels in schizophrenic
patients, whereas they found it unaffected in patients with
0
100
500
550
600
650
700
750
800
Plasma Cortisol
psychosocial vs. physical stress
-2 +2
+15 +30 +40
Time (min)
Plasma Cortisol (nmol/l)
psychosocial stress
physical stress
(A)
STRESS
TEST
*** ***
**
Salivary Cortisol
psychosocial vs. physical stress
0
1
12
15
18
21
24
27
30
-2 +2
+15 +30 +40
Time (min)
Salivary Cortisol (nmol/l)
psychosocial stress
physical stress
STRESS
TEST
(B)
***
***
0
100
200
300
500
1500
2500
3500
Noradrenaline
psychosocial vs. physical stress
-2 +2
+15 +30 +40
Time (min)
Noradrenaline (pg/ml)
psychosocial stress
physical stress
(C)
STRESS
TEST
***
***
***
**
*
0
10
20
30
40
50
200
400
600
Adrenaline
psychosocial vs. physical stress
-2 +2 +15 +30 +40
Time (min)
Adrenaline (pg/ml)
psychosocial stress
physical stress
(D)
STRESS
TEST
***
***
*** **
Fig. 2 Hormone levels before and after psychosocial and physical stress conditions. a–dProgression of stress hormones in plasma and saliva
before and after psychosocial and physical stress are shown. aPlasma cortisol is plotted in nmol/l over time. Both stress conditions led to an increase
in plasma cortisol with the highest values measured 15 min after cessation of psychosocial and physical stress. Plasma cortisol increased by 107 nmol/
l after psychosocial stress and by more than 210 nmol/l after physical exhaustion (main effect time: F
(1.96, 74.48)
=25.26, p< 0.001, η
2
=0.399; main
effect condition: F
(1, 38)
=4.57, p=0.039, η
2
=0.107; time × condition interaction effect: F
(1.96, 74.48)
=13.50, p< 0.001, η
2
=0.262). bIncreased salivary
cortisol (in nmol/l) in psychosocial and physical stress conditions. Cortisol levels after psychosocial stress peaked after 15 min, whereas they increased
until 30 min after physical stress (main effect time: F
(1.83, 69.58)
=17.47, p< 0.001, η
2
=0.315; main effect condition: F
(1, 38)
=2.20, p=0.146, η
2
=0.055;
time × condition interaction effect: F
(1.83, 69.58)
=11.10, p< 0.001, η
2
=0.226). Psychosocial and physical stress led to an increase of noradrenaline (c)
and adrenaline (d). Immediately after psychosocial stress, the concentration of noradrenaline and adrenaline doubled before dropping back to
baseline levels within 15 min. Physical stress led to an 18-fold increase of noradrenaline (c) and adrenaline (d) (noradrenaline: main effect time: F
(2.21,
83.89)
=226.57, p< 0.001, η
2
=0.856; main effect condition: F
(1, 38)
=152.10, p< 0.001, η
2
=0.800; time × condition interaction effect: F
(2.21, 83.89)
=
104.94, p< 0.001, η
2
=0.734; adrenaline: main effect time: F
(2.21, 73.04) =
103.06, p< 0.001, η
2
=0.757; main effect condition: F
(1, 33)
=48.54, p< 0.001,
η
2
=0.595; time × condition interaction effect: F
(2.21, 73.04)
=55.80, p< 0.001, η
2
=0.628). Values are reported as means ± SEM. The data were analyzed
with repeated measures ANOVAs. In case of significant effects, post-hoc test was carried out to check for group differences (*p≤0.05,
**p≤0.01 ***p≤0.001)
Hummel et al. Translational Psychiatry (2018) 8:236 Page 7 of 10
mood disorders. These inconsistent results might in part be
due to technical confounders in the quantification of cf-
mtDNA fragments, including extraction methods and
ampliconsize.IncontrasttoextracellulargenomicDNA,
which is at least partially protected from enzymatic degra-
dation by nucleosomal packaging, mtDNA when freely
exposed to blood plasma is highly vulnerable to complete
degradation by serum DNase I. Cf-mtDNA will thus be
rapidly degraded to fragment sizes below the detection
limits of PCR-based methods, as recently shown in plasma
from sepsis patients
41
. Exercise-triggered transient rise in
serum DNase I activity will further speed up this process
under physical stress conditions
7
. Thus, it cannot be
excluded that we and others underestimated the true
release kinetics of mtDNA under various settings
6,42,43
,
which might also explain the lack of correlation between
cortisol and catecholamines and cf-mtDNA and highlight
the importance of standard plasma DNA detection
procedures.
In response to the TSST, quantity of plasma cfDNA and
cf-mtDNA levels doubled, peaking immediately after the
end of stress exposure. We addressed the question of
whether there might be a stress-specific cfDNA signature
by comparing cfDNA increases, fragment length, and
DNA methylation as an indicator of cellular origin
between psychosocial stress exposure and physical stress
in the same individuals. Following strenuous physical
exercise, we found a fivefold increase in cfDNA after
cessation of exercise, as previously shown by Beiter et al.
6
.
Furthermore, fragment analysis of cfDNA revealed a dis-
tinct fragmentation pattern of stress-provoked cfDNA
levels, with physical strain inducing cfDNA molecules of
smaller sizes.
In addition to cfDNA increase and alterations in the
number of small fragments, we observed divergent pat-
terns of DNA methylation over time in the CpG island of
our reporter gene’s(HOXA5) promoter when comparing
both stress paradigms (Fig. 1c). There was a shift towards
lower cfDNA methylation after psychosocial compared to
increased methylation after physical stress. CfDNA which
leads to lowered total methylation of cfDNA must derive
from cells with a hypomethylated HOXA5 gene locus,
whereas cfDNA that increases total DNA methylation of
plasma cfDNA at that specific genomic locus must derive
from cells with a hypermethylated HOXA5 locus.
According to various databases (blueprint-epigenome,
University of California, Santa Cruz (UCSC) genome
browser), the HOXA5 gene locus is hypomethylated in
brain and neuronal cells and shows hypermethylation in
blood and muscle cells. The exact cellular origin remains
elusive, but these results do point towards different cel-
lular origins of cfDNA when triggered by psychosocial
stress compared to physical exercise, suggesting that the
release of cfDNA might not be just disposal of damaged
DNA but rather a result of a regulated process.
Whereas we could show differences in quantity and
quality between the two conditions, our investigation did
not address whether differences in cfDNA signatures
between conditions might also reflect differences in
function. As a comprehensive understanding of molecular
processes involved in the release and function of cfDNA is
still lacking, we can only speculate about stimulus-specific
downstream effects of different cfDNA populations and
release mechanisms at this point. Interestingly,
active release of DNA, either from nuclear or mitochon-
drial genomic content, has recently emerged as a
remarkable feature of several white blood cell lineages.
Moreover, it has been shown that leukocyte-derived DNA
may play a crucial role in the regulation of immune
responses and thus may serve as another universal type of
communication mechanism to shape immunity at multi-
ple levels
25,26
.
010203040
0
1×10
2×10
3×10
6×10
9×10
1.2×10
Correlation Salivary Cortisol and cfDNA
Salivary Cortisol Delta (nmol/l)
cfDNA Delta (copies/ml)
psychosocial stress
physical stress
R= .291, p = .014
R= .082, p = .221
(A)
0 100 200 300 400 500
0
1
×
10
2
×
10
3
×
10
6
×
10
9
×
10
1.2
×
10
Correlation Plasma Cortisol and cfDNA
Plasma Cortisol Delta (nmol/l)
cfDNA Delta (copies/ml)
psychosocial stress
physical stress
R= .176, p = .0 66
R= .080, p = .2 28
(B)
Fig. 3 a A significant positive correlation between salivary cortisol increases and cfDNA increases (peak minus baseline) was observed in the physical
stress condition. The association between increases in salivary cortisol and cfDNA levels after the TSST followed the same direction but was not
statistically significant. bA trend toward significance for a positive correlation between plasma cortisol and cfDNA increase after physical stress and a
similar, non-significant relationship for the TSST was observed
Hummel et al. Translational Psychiatry (2018) 8:236 Page 8 of 10
Quick release of cfDNA after psychosocial and physical
stress as observed might possibly be attributed to vital
NETotic processes rather than to slow processes such as
apoptosis and necrosis, raising the possibility that stress-
induced cfDNA release might be involved in stress-
associated immune system regulation
44
. NETs have
emerged as an important and highly conserved innate
host defense mechanism and, recently, netting neu-
trophils have also been observed to occur in the blood in
response to strenuous exercise
7
. On the other hand,
aberrant or unresolved release of NETs has been docu-
mented to contribute to the pathogenesis of diverse auto-
inflammatory conditions, vascular inflammation, throm-
bosis, and cancer
45–48
. However, the proportion to which
NETs may contribute to increased cfDNA levels in dif-
ferent conditions is currently unknown.
Another possible source of DNA in the circulation after
different stress conditions are extracellular vesicles (EVs).
Following appropriate stimuli, most cells release EVs
which carry RNA, lipids, and DNA
49–53
at least on their
surface
54
. Interestingly, Lutgendorf et al.
55
recently
reported that social support influences the RNA popula-
tion of exosomes, associated with improved health out-
comes in patients with ovarian carcinoma. Further studies
are warranted to identify to which extent EVs contribute
to cfDNA increases following stress, and in addition, how
EV-specific DNA and RNA populations might influence
stress-specific aspects of human behavior and health
outcomes.
As a limitation, it needs to be noted that only males
were investigated, and that the sample size was modest.
Furthermore, it cannot be entirely excluded that the stress
associated with catheter placement was responsible for
cfDNA increases. However, we believe that this is extre-
mely unlikely, as the catheter was inserted 45 min before
testing which is common practice when potential effects
of blood draw should be avoided. Should venipuncture
lead to cfDNA release, most of this elevation would have
already metabolized before start of the stress test, as
cfDNA has a half-life of about 15 min. Furthermore,
cfDNA values 2 min before testing were low and highly
similar between conditions, with differences emerging
after stimulation.
Human, animal, and cell culture studies suggest that
cfDNA can significantly influence the physiological
activity of intact living cells (reviewed in ref.
56
). Although
the underlying mechanisms are poorly understood as of
yet, it is evident that regular physical exercise provides
ample protection from dysregulation of immune home-
ostasis that compromises the body’s defense systems, as
observed in multiple chronic disorders
57–60
. On the other
hand, social and psychosocial stressors have a profound
negative impact on proper immune system balance and
can lead to mood disorders, such as depression
61
.
Necessarily, a better understanding of the acute
stress response pattern in health and disease and its dif-
ferential outcomes is inevitable in facing the epidemic
increase in lifestyle-associated physical as well as
mental illnesses
57–60,62,63
.
In summary, we could show that psychosocial stress
exposure as well as physical exercise lead to increased
cfDNA release, with stimulus and/or intensity-dependent
differences in magnitude, size, and methylation pattern
following different challenge protocols. Our findings
support the idea of using cfDNA as a biomarker in
experimental stress research in addition to hormone levels
such as cortisol or catecholamines. Furthermore, cfDNA
could possibly be used for diagnosis or monitoring of
treatment progression in stress-related mental disorders
and for subgrouping of patients with similarities in stress-
related pathophysiological processes. However, studies
clarifying the functional physiological role of cfDNA are
warranted.
Acknowledgements
The authors thank Annika Mühlenkamp for her expert technical assistance. We
further acknowledge support by the DFG Open Access Publication Funds of
the Ruhr-Universität Bochum.
Author details
1
Department of Genetic Psychology, Faculty of Psychology, Ruhr-University
Bochum, Universitätsstraße 150, 44801 Bochum, Germany.
2
Department of
Sports Medicine, Medical Clinic, Eberhard-Karls-University of Tübingen, Otfried-
Müller-Straße 10, 72076 Tübingen, Germany.
3
Department of Sports Medicine
& Sports Nutrition, Faculty of Sport Science, Ruhr-University Bochum,
Universitätsstraße 150, 44801 Bochum, Germany.
4
Department of Cognitive
Psychology, Faculty of Psychology, Ruhr-University Bochum, Universitätsstraße
150, 44801 Bochum, Germany.
5
Institute for Transfusion Medicine, University
Hospital Essen, University Duisburg‑Essen, Hufelandstraße 55, 45122 Essen,
Germany
Conflict of interest
The authors declare that they have no conflict of interest.
Publisher's note
Springer Nature remains neutral with regard to jurisdictional claims in
published maps and institutional affiliations.
Supplementary Information accompanies this paper at (https://doi.org/
10.1038/s41398-018-0264-x).
Received: 8 May 2018 Revised: 2 August 2018 Accepted: 7 September 2018
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