Neutrophils release extracellular DNA traps in

response to exercise
Thomas Beiter, Annunziata Fragasso, Jens Hudemann, Marius Schild, Jürgen
Steinacker, Frank C. Mooren and Andreas M. Niess
J Appl Physiol 117:325-333, 2014. First published 15 May 2014; doi:10.1152/japplphysiol.00173.2014

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Direct measurement of cell-free DNA from serially collected capillary plasma during
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Sarah Breitbach, Björn Sterzing, Carlos Magallanes, Suzan Tug and Perikles Simon
J Appl Physiol, July 15, 2014; 117 (2): 119-130.
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Neutrophils release extracellular DNA traps in response to exercise
Thomas Beiter,1 Annunziata Fragasso,1 Jens Hudemann,1 Marius Schild,2 Jürgen Steinacker,3
Frank C. Mooren,2 and Andreas M. Niess1
1
Department of Sports Medicine, Medical Clinic, Eberhard-Karls-University of Tuebingen; 2Department of Sports Medicine,
Justus-Liebig University, Giessen; and 3Division of Sports and Rehabilitation Medicine, Ulm University Medical
Center, Germany
Submitted 21 February 2014; accepted in final form 8 May 2014
Beiter T, Fragasso A, Hudemann J, Schild M, Steinacker J,
Mooren FC, Niess AM. Neutrophils release extracellular DNA traps
in response to exercise. J Appl Physiol 117: 325–333, 2014. First
published May 15, 2014; doi:10.1152/japplphysiol.00173.2014.—In-
tense exercise evokes a rapid and transient increase in circulating
cell-free DNA (cf-DNA), a phenomenon that is commonly observed
in a variety of acute and chronic inflammatory conditions. In this
study, we aimed to shed new light on the release and clearance
mechanisms of cf-DNA in response to exercise. We hypothesized that
activated neutrophils may primarily contribute to exercise-evoked
cf-DNA levels by releasing neutrophil extracellular traps (NETs).
Analysis of plasma and/or serum samples from male athletes at rest
and in response to exhaustive treadmill exercise revealed an immedi-
ate and transient increase in cf-DNA that was concomitantly counter-
balanced by an increase in serum DNase activity. Consistently, rapid
release and clearance kinetics for cf-DNA could also be observed in
response to intensive cycling exercise, with no significant differences
between endurance-trained (V̇O2max ⬎57 ml·min⫺1·kg⫺1) and healthy
(V̇O2max ⬍49 ml·min⫺1·kg⫺1) sedentary individuals. In postexercise
blood smear samples, we detected seemingly intact neutrophils dis-
playing morphological signs of NET release, as indicated by abnormal
swollen nuclei and emanating DNA fibers. In support, we observed a
striking correlation of postexercise cf-DNA concentrations with
plasma levels of the granule-derived enzyme myeloperoxidase. Our
study indicates that intense exercise induces liberation of NETs,
which is sufficiently counterbalanced in healthy individuals by a
concomitant rise in serum DNase activity. As aberrant release of
NETs has been linked to diverse disease states, monitoring of cf-
DNA/DNase levels or activities in response to standardized exercise
testing could provide a valuable tool to identify people who are at
increased risk for cardiac ischemia, thrombosis, autoimmunity, or
chronic fatigue.
neutrophil extracellular traps; cell-free DNA; DNase
AT THE ADVENT OF THE 21st century, the prevalence of chronic
diseases in developed and developing countries is increasing at
an alarming rate (37). Chronic conditions like obesity, diabe-
tes, and cardiovascular disease contribute largely to morbidity
and mortality all over the globe and place increasing pressure
on health care systems (4). Because chronic disorders are
primarily attributable to lifestyle-related factors, a sizable frac-
tion of this disease burden could substantially be minimized by
nonpharmacological interventions.
Adopting a regular exercise routine is widely recognized as
offering protection against obesity, insulin resistance, and ath-
erosclerosis (28, 57). Moreover, there is accumulating knowl-
edge that exercise intervention provides a true treatment mo-
Address for reprint requests and other correspondence: T. Beiter, Dept. of
Sports Medicine, Medical Clinic, Eberhard-Karls-Univ. of Tuebingen, Hoppe-
Seyler-Str. 6, 72076 Tuebingen, Germany (e-mail: thomas.beiter@med.uni-
tuebingen.de).
http://www.jappl.org 8750-7587/14 Copyright © 2014 the American Physiological Society
J Appl Physiol 117: 325–333, 2014.
First published May 15, 2014; doi:10.1152/japplphysiol.00173.2014.
dality in a wide variety of chronic inflammatory conditions (28,
57). However, if we indeed consider exercise as a discrete
therapeutic modality, we also have to consider that—as with
any medical treatment—prescription of exercise mode, inten-
sity, and duration must be based on a careful evaluation of risks
and benefits, taking into account possible contraindications and
the patient’s individual risk profile. To develop appropriate
exercise intervention guidelines, it is inevitable to reach a
deeper understanding of how, when, and why physical activity
interferes with basic mechanisms of our immune system.
Depending on intensity and duration, acute physical exercise
constitutes a multiple-stressor situation involving oxidative,
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metabolic, mechanical, thermal, and neurohormonal stressors
to trigger mobilization and activation of leucocytes, acute
phase proteins, platelet activation, and alterations of throm-
botic and fibrinolytic pathways (58). Recently, it has been
shown that strenuous exercise likewise provokes an immediate
and transient increase in plasma circulating cell-free DNA
(cf-DNA) (2, 5, 22). Increased concentrations of cf-DNA have
for a long time been implicated in a wide range of acute and
chronic inflammatory conditions, including cancer, autoim-
mune diseases, trauma, sepsis, stroke, and myocardial infarc-
tion (54). As a consequence, cf-DNA is considered to have
diagnostic and prognostic power in malignant as well as in
nonmalignant diseases (32). Remarkably, despite intensive
research over the course of decades, there still is no consensus
on the origin, release mechanisms, and biological significance
of cf-DNA. Most commonly, the presence of cf-DNA in the
circulation has been ascribed to cell death, either directly by
release from necrotic and apoptotic cells, or indirectly by
liberation from macrophages or scavengers following absorp-
tion of dying cells (54).
A novel fascinating explanation of how DNA can actively be
released under inflammatory conditions has recently become
apparent by the discovery of an evolutionary highly conserved
first-line defense mechanism that allows neutrophils to expel
their DNA in response to infectious and endogenous stimuli,
thereby forming an antimicrobial meshwork of chromatin and
granule proteins, termed neutrophil extracellular traps (NETs)
(8). Accumulating evidence indicates that formation of NETs
plays a pivotal role in innate immune responses, allowing
neutrophils to capture, neutralize, and degrade a variety of
invading microorganisms (9). Moreover, NETs have been
shown to constitute a scaffold and stimulus for platelet and red
blood cell adhesion, fibrin deposition, and thrombus formation,
thereby providing a physical barrier to prevent microbial dis-
semination (12, 25, 40). Aside from infection, NETs have also
been found to be implicated in sterile inflammation, and,
besides their beneficial properties, dysregulated NET forma-
tion emerges to profoundly contribute to the pathogenesis of
325
326 NETs in Exercise
diverse autoinflammatory conditions, vascular inflammation,
and atherogenesis (19, 41, 48, 53).
By monitoring the time course of cf-DNA plasma concen-
trations in healthy individuals during and after incremental
treadmill running, we recently reported rapid release and clear-
ance characteristics of cf-DNA in response to exhaustive ex-
ercise (5). Based on this observation, we hypothesized that the
immediate exercise-induced increase in cf-DNA might be in-
dicative of NETs that could be formed within the vasculature,
as has recently been observed in septic blood (12, 48, 59). We
further speculated that release of cf-DNA in response to acute
exercise might be efficiently counterbalanced in healthy indi-
viduals by an increase in serum DNase 1 activity. To test our
hypothesis, we measured cf-DNA concentrations as well as
DNase 1 activity in the blood of healthy individuals at rest and
in response to intensive endurance exercise. As regular phys-
ical activity is generally considered to exert beneficial effects
on immune functions, we included sedentary as well as well-
trained individuals in our study. In addition, we quantified
plasma markers of inflammation, muscle injury, and neutrophil
activation. We further attempted to visualize exercise-induced
NET formation in the bloodstream by immunocytochemical
staining of blood smear samples. Our data indicate that intra-
vascular NETs are released during intensive exercise.
MATERIALS AND METHODS
Study design. In the first part of the study, we attempted to provide
initial evidence for the proposed release and clearance mechanisms of
cf-DNA. We chose an exhaustive treadmill protocol that has recently
been shown as suitable for provoking immediate and pronounced
increases in cf-DNA (5). The second part of the study aimed to
confirm these results in a different exercise setting that allowed
assessment of whether the outcome variables would be influenced by
the training status. All experimental procedures were approved by the
Institutional Review Board of the University of Tuebingen according
to the Declaration of Helsinki.
Study participants and exercise setups. All subjects were fully
informed of the experimental protocols and gave written informed
consent before the study commenced. Vital signs, anthropometric
measurements, and physical examinations were conducted to evaluate
health status. Subjects were excluded if they presented with any acute
or chronic disease, major or minor injuries, or use of prescribed
medication. In the initial study part, six well-trained male athletes
(median age, 24 yr; range, 20 –33 yr) who were rerecruited from our
previous study (5) repeated a graded exercise test until volitional
exhaustion on a motorized treadmill (Saturn, HP COSMOS, Traun-
stein, Germany) at a constant incline of 1% with an increment of 2
km/h every 3 min. Serum and EDTA blood samples were taken before
(pre), immediately after (post), and 30 min (⫹30 min) after the run.
To determine effects of training status, healthy male subjects were
recruited who were either engaged in regular endurance exercise of
more than 5 h/wk or habitually spent less than 2 h/wk with physical
activity. All subjects underwent preliminary baseline screening in-
cluding lactate diagnostic and maximal bicycle spiroergometry to
determine maximal oxygen uptake (V̇O2max) as defined by the highest
oxygen uptake rate measured during the test, and expressed as
milliliters of oxygen used in one minute per kilogram of body weight
(ml·min⫺1·kg body wt⫺1). The power (in watts) performed at the time
of 100%, 80%, and 60% V̇O2max was recorded. Depending on their
endurance capacity, six sedentary or untrained individuals (UT) and
six endurance-trained individuals (ET) who met the final inclusion
criteria of V̇O2max ⬍49 ml·min⫺1·kg body wt⫺1 for the UT group and
V̇O2max ⬎57 ml·min⫺1·kg body wt⫺1 for the ET group, respectively,
were included to take part in the study. The anthropometric data for
J Appl Physiol • doi:10.1152/japplphysiol.00173.2014 • www.jappl.org
• Beiter T et al.
both groups are shown in Table 1. Within 4 wk from the initial
performance diagnostic, the study participants performed the main
exercise protocol that consisted of 60 min of high intensity cycling
exercise on a cycle ergometer (Ergometrics 800 S, Ergoline, Bitz,
Germany) at a power requiring 80% of the V̇O2max (including 10 min
of warm-up cycling at 60% V̇O2max). All exercise sessions were
conducted in the morning (9:00 AM) after an overnight fast and after
receiving a standardized breakfast (8:00 AM). Serum and EDTA
blood samples were collected at rest (pre), immediately after (post),
and 3 h (⫹3 h) after the cycling exercise protocol. Routine blood
variables were determined for each time point by standard methods.
Blood samples were centrifuged at 1,600 g for 10 min, followed by
5 min of centrifugation at 16,000 g, and aliquots were stored at ⫺80°C
until further analysis.
Quantification of cf-DNA. DNA was extracted from 400 ␮l of
plasma according to the blood and body fluid protocol of the QIAamp
Blood Mini Kit (Qiagen, Hilden, Germany) with a final elution
volume of 100 ␮l. cf-DNA equivalents were quantified by SYBR
green real-time PCR that targeted an 88-bp fragment of the chromo-
somal myostatin (MSTN) gene locus, as described previously (5).
Briefly, samples were run in triplicates on an iCycler single-color
detection system (BioRad, Munich, Germany) using the QuantiFast
SYBR Green PCR Kit (Qiagen). DNA levels were calculated from a
standard curve generated from serial dilutions of a genomic calibrator
by plotting the threshold cycle value against the logarithm of calibra-
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tor copy numbers. Copies of the target sequence per plasma sample
volume were then calculated as described by Chiu et al. (11), and total
quantities of cf-DNA are given as genome equivalents (GE) per
milliliter of plasma, with one GE being defined as the amount of the
target sequence contained in a single haploid cell. The lower quanti-
fication and detection limits of the method were reproducibly identi-
fied at 500 GE/ml and 250 GE/ml plasma, respectively.
Quantification of serum DNase 1 activity. DNase activity in serum
samples was determined by using Org 590 DNase activity ELISA
(Orgentec, Mainz, Germany) according to the manufacturer’s instruc-
tions. This assay measures the intrinsic DNA degradation capacity of
a serum sample by the turnover of an immobilized DNase substrate
coated on the chamber surfaces of a microwell plate. Remaining
substrate is quantified photometrically at 450 nm, and the amount of
color reaction is inversely proportional to the DNase activity in the
serum sample (activity reduction). To make data more amenable for
quantitative comparison, we included a standard curve [5.31 up to
277.53 milli-Kunitz units (mKuU)/ml] with defined activities of a
recombinant human DNase 1 standard provided by the manufacturer.
Staining of blood smears. Blood droplets (35 ␮l) from EDTA
whole blood samples were placed on glass slides and gently spread
using the edge of a second glass slide. For histological staining, blood
smears were air-dried, fixed in methanol, and stained by standard
Giemsa stain (Carl Roth, Karlsruhe, Germany) procedure.
For immunohistochemical staining of neutrophils, blood smears
were fixed in 4% paraformaldehyde in PBS for 2 h at room temper-
ature, rinsed with PBS, permeabilized with 0.5% Triton X-100 PBS
for 1 min, and blocked overnight in 2% BSA PBS at 4°C. Incubation
Table 1. Physical characteristics of endurance-trained
and untrained participants
V̇O2max, Power Output at 80%
Age, yr BMI, kg/m2 ml 䡠 min⫺1 䡠 kg V̇O2max, Watt
ET 27.0 22.7 65.4 250
(n ⫽ 6) (26–30) (21–24) (58–67)* (230–310)*
UT 26.5 21.9 39.5 150
(n ⫽ 6) (19–28) (20–25) (30–48) (50–190)
Values are median (range); BMI, body mass index; V̇O2max, maximal
aerobic capacity; ET, endurance trained; UT, untrained. *P ⬍ 0.01, signifi-
cantly different compared with UT group.
 NETs in Exercise • Beiter T et al. 327
with primary rabbit anti-human neutrophil elastase antibodies 15.2-fold, from 45.1 (29.4 –79.4) mKuU/ml at rest to 310.9
(ab68672, 1:200; Abcam, Cambridge, United Kingdom) or the corre- (187.6 –594.3) mKuU/ml (P ⫽ 0.005) postexercise (Fig. 1B).
sponding negative control antibodies (Ab 46540, Abcam) was per- Increased serum enzyme activity was of a transient nature and
formed for 1 h at room temperature in 2% BSA PBS, followed by
decreased to preexercise levels at 30 min after cessation of
secondary staining with Cy3-conjugated donkey anti-rabbit IgG (711-
166-152, 1:400, Dianova, Hamburg, Germany) for 1 h at room exercise (median 47.7 mKuU/ml; range 33.0 –194.2).
temperature. DNA was labeled with 4,6-diamidino-2-phenylindole Postexercise blood smears reveal NET-like structures. Con-
(1.25 ␮g/ml; Invitrogen, Life Technologies, Darmstadt, Germany). sidering the rapid appearance of excess cf-DNA in the circu-
Specimens were mounted with Vectashield (Vector Laboratories, lation of exercising subjects, we analyzed Giemsa-stained
Peterborough, United Kingdom), and neutrophils were visualized by blood smears from pre- and postexercise blood samples to
immunofluorescence microscopy (Axiovert, Zeiss, Goettingen, Ger- screen for suspicious signs of damaged or dying blood cells. In
many). peripheral blood smear slides prepared immediately after the
Quantification of plasma markers. Quantification of 89 inflamma-
tion-associated proteins in plasma samples was performed at Rules
exhaustive treadmill run, we observed the scattered but striking
Based Medicine (Myriad RBM, Austin, TX) using their human MAP appearance of seemingly disintegrated cells with a decon-
v1.6 panel of Luminex-based multiplex assays as described by the densed chromatin structure (Fig. 2A). Similar structures are
vendor. Analytes containing values below the lower detection limit apparent in blood smears from patients with malaria (3), and
were excluded from analysis. have recently been documented in blood smears from stored
Plasma volume changes. Study participants were instructed to red blood cell units (24). Here, these cells were recognized as
drink water at two defined time points during the cycling exercise circulating neutrophils that are releasing extracellular DNA
protocol. To confidently exclude biased measures due to plasma
webs, termed NETs. To obtain proof of principle, we used
volume shift, changes in plasma concentrations were calculated from
hemoglobin and hematocrit values and concentrations of postexercise immunocytochemistry to visualize intravascular neutrophils by
variables were corrected according to Kraemer and Brown (35). For neutrophil elastase staining and to clearly characterize NET-
all analyzed postexercise samples, calculated losses in plasma volume like structures in postexercise blood smears. As shown in Fig.

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were below 6% (range: 1.5% to 5.4%). 2, suspicious NET-like structures emerged as seemingly intact
Statistical analysis. Statistical analysis was performed using JMP neutrophils with abnormal swollen nuclei and dispersed nu-
(version 10; SAS Institute, Cary, NC) and SPSS statistical software clear staining patterns that were surrounded by a delicate
(version 15; SPSS, Chicago, IL). Differences within groups over time network of emanating DNA (Fig. 2B). Control stainings of
were analyzed by Friedman’s two-way nonparametric ANOVA, fol-
lowed by Wilcoxon’s matched-pairs signed-rank test. The Mann-
isolated neutrophils treated with phorbol myristate acetate
Whitney U-test was used to compare between group variables. Spear- (PMA; data not shown) exemplified that the observed pheno-
man rank correlation coefficients (CCs) were calculated with Bonfer- type is clearly distinct from the commonly described lytic
roni-adjusted significance levels to estimate the correlation between mechanism of NET release that culminates in the rupture of the
variables. A value of P ⬍ 0.05 was regarded as statistically signifi- cellular membrane and the concomitant expulsion of filamen-
cant. Data are presented as median (range). tous chromatin structures (8), but could rather be attributed to
the recently discovered rapid mechanism of vesicular-mediated
RESULTS
NET release (48).
Release of cf-DNA in response to acute exercise is counter- Trained and untrained healthy individuals do not differ in
balanced by serum DNase 1. An immediate, pronounced, and their cf-DNA/DNase responses to exercise. Because aberrant
transient increase in cf-DNA levels could be observed in six release of cf-DNA/NETs has been implicated in diverse
healthy young athletes in response to an acute bout of exhaus- chronic inflammatory conditions, we wanted to address
tive treadmill exercise, rising by 14.9-fold from median base- whether sedentary (untrained, UT) and habitually exercising
line values of 1,645 (1,262– 4,527) GE/ml at rest to 25,511 (endurance-trained, ET) individuals display distinct cf-DNA/
(10,673–54,383) GE/ml (P ⫽ 0.005) at the end of the run, and DNase responses to acute exercise. To achieve comparable
declining to 4.1-fold baseline concentrations after 30 min of workload and duration for both groups, we chose a 1-h high-
recovery (median 7,718 GE/ml; range 4,156 –16,730; P ⫽ intensity cycling protocol adjusted to the individual V̇O2max
0.008; Fig. 1A). Rapid clearance kinetics of cf-DNA could be level. Both groups did not differ in their baseline cf-DNA
attributed to an immediate rise in serum DNase 1 activity by levels, with a median plasma concentration of 2,078 (638 –

A B
60000 ** ** 700 ** **
Plasma DNase activity (mKuU/ml)

50000 600
Plasma cf-DNA (GE/ml)

Fig. 1. Values of cell-free DNA (cf-DNA)

500 plasma concentrations (A) and serum DNase 1
40000 activity (B) in the blood of well-trained individ-
400 uals (n ⫽ 6), determined at rest (pre), in re-
30000 sponse to exhaustive treadmill exercise (post),
300
and after 30 min of recovery (⫹30 min). Bar,
20000 median of the group. **P ⱕ 0.01, significant
200
difference between sampling points.
10000 100

0 0
pre post +30 min pre post +30 min

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328 NETs in Exercise • Beiter T et al.

4,197) GE/ml in the ET group and 1,582 (1,240 –3,465) GE/ml
in the UT group (Fig. 3A). Likewise, there was no difference in
postexercise levels as both groups showed an equally pro-
nounced increase in cf-DNA, rising significantly by 5.9-fold in
the ET group to 9,425 (5,765–17,549) GE/ml (P ⫽ 0.004) and
by 5.8-fold in the UT group to 10,934 (7,525–18,774) GE/ml
(P ⫽ 0.004), respectively. Three hours after cessation of
exercise, cf-DNA plasma concentrations in both the ET group
(median 2,948 GE/ml; range 1,515– 8,489) as well as in the UT
group (median 2,131 GE/ml; range 1,376 –3,673) had returned
to preexercise levels. The same pattern was visible for serum
DNase 1 activity (Fig. 3B). Significant enhanced activity by
8.1-fold and 7.1-fold, respectively, could be observed in re-
sponse to exercise for both the ET group (P ⫽ 0.004) as well
as the UT group (P ⫽ 0.002), with median baseline values of
19.3 (13.5–23.6) mKuU/ml and 19.2 (17.4 –25.9) mKuU/ml,
respectively, and median postexercise values of 143.6 (122.9 –
296.3) mKuU/ml and 133.3 (56.8 –198.1) mKuU/ml, respec-
tively. A decline to near baseline activity was consistently
observed in both groups after 3 h of recovery, with median
serum activities of 23.0 (14.1–97.2) mKuU/ml and 30.3 (25.9 –
130.1) mKuU/ml respectively.
exercise (Spearman’s CC ⫽ 0.699, P ⫽ 0.011; Fig. 3E). Of
note, no significant association could be observed with the
muscle damage marker myoglobin (Spearman’s CC ⫽ ⫺0.028,
P ⫽ 0.931; Fig. 3F). Pronounced liberation of myoglobin
became apparent at 3 h after cessation of exercise (Fig. 3D),
with a significant correlation to FABP plasma levels (Spear-
man’s CC ⫽ 0.937, P ⬍ 0.0001), another early cytoplasmic
muscle damage marker. Because MPO is particularly abundant
in the primary granules of neutrophils, correlation of cf-DNA
and MPO levels in postexercise samples further supports the
idea that netting neutrophils might substantially contribute to
the immediate cf-DNA release in response to exercise.
DISCUSSION
Increased concentrations of cf-DNA are frequently observed
in acute and chronic inflammatory conditions (54). While the
prognostic and diagnostic value of this phenomenon has been
extensively attributed in diverse malignancies as well as in
infectious and autoinflammatory diseases (32), the biological
significance of nonpathological release of cf-DNA in the
course of physical exercise remains enigmatic. It has initially

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Release of cf-DNA in response to exercise is associated with
increased plasma myeloperoxidase levels. To further charac-
terize the inflammatory milieu of exercise-induced release of
cf-DNA, we determined plasma concentrations of 89 inflam-
mation-associated proteins using a multiplex quantification
approach. The analytes that could be found significantly in-
creased in the circulation of either group at any of the sampling
points following exercise were the interleukins IL-6, IL-8,
IL-10, and IL-16; growth hormone; the secretable matrix
metalloproteinases MMP2, MMP3, and MMP9; the myeloid
cell-specific enzyme myeloperoxidase (MPO; Fig. 3E); as well
as myoglobin (Fig. 3D) and the heart-type fatty acid-binding
protein (FABP), both representing cytoplasmic muscle constit-
uents (Table 2). Correlation analysis revealed that MPO was
the only analyte with postexercise plasma levels significantly
correlated with the amount of cf-DNA released in response to
been assumed that leakage of nuclear content from traumatized
muscle fibers may primarily account for exercise-induced cf-
DNA levels, thus simply representing a bystander marker of
muscle damage (2). This view has recently been challenged by
thorough time-course analyses, showing clearly distinct release
kinetics of cf-DNA and cytosolic markers of muscle damage
during and following strenuous endurance exercise (5, 23).
Here, we provide evidence that release of cf-DNA in response
to exercise rather reflects an active effector mechanism of
innate immunity, namely the formation of NETs by peripheral
blood neutrophils. Our results show that 1) exercise-induced
cf-DNA is efficiently counterbalanced in healthy individuals
by a concomitant increase in serum DNase 1 activity; 2) the
immediate appearance of cf-DNA in the circulation clearly
precedes and is not correlated with the early muscle damage

Fig. 2. Representative images of neutrophil extra-

cellular trap (NET)-like structures in the postexer-
cise blood of a well-trained subject. Blood smears
were prepared immediately after exhaustive tread- A B
mill exercise and Giemsa stained for cytological NE NE NE
examination (A). Intact neutrophils are clearly de-
lineated by their lobular and segmented nuclei (A,
overview image and top left). NET-like structures
appear as seemingly disintegrated cells with a
decondensed chromatin structure and fibrous pro-
trusions of DNA (A, overview image and top DAPI DAPI DAPI
right). Blood smears were further analyzed by
immunocytochemistry for neutrophil elastase
(NE; B). Bottom row panels show merged immu-
nofluorescence images of representative neutro-
phils in postexercise blood smears. NE is shown in
red (top row). DNA was labeled with 4,6-di-
amidino-2-phenylindole (DAPI) and is shown in Merge Merge Merge
blue (middle row). Intact neutrophils were identi-
fied as NE-positive cells with typical segmented
nuclei (B, left). Netting neutrophils were charac-
terized as NE-positive cells with seemingly intact
membrane structure and disintegrated nuclei (B,
middle and right columns), and NET-like struc-
tures were identified as delicate networks of ex-
tracellular DNA. Bar, 10 ␮m.

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 NETs in Exercise • Beiter T et al. 329

A ** ** B **
20000 ** * ** *
350

Plasma DNase activity (mKuU/ml)

300
Plasma cf-DNA (GE/ml)

15000
250

200
10000
150

5000 100

50

0 0
ET UT ET UT ET UT ET UT ET UT ET UT
pre post +3h pre post +3h Fig. 3. Effect of acute exercise and training
status on plasma levels of cf-DNA (A), serum

C ** D ** DNase activity (B), plasma concentrations of

myeloperoxidase (MPO; C), and myoglobin
300 * 1000 ** (D). Values were retrieved from blood samples
of endurance-trained (ET; n ⫽ 6; closed circles;
Plasma myoglobin (ng/ml)

250 800 V̇O2max ⬎57 ml·min⫺1·kg body wt⫺1) and sed-

Plasma MPO (ng/ml)

entary (untrained, UT; open circles; n ⫽ 6;

200 V̇O2max ⬍49 ml·min⫺1·kg body wt⫺1) individ-
600
uals at rest (pre), after 60 min of intense cycling

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150 exercise (post), and after 3 h of recovery (⫹3
400 h). Bar, median of the group. A significant
100 positive association between plasma cf-DNA
200 levels and MPO concentrations could be de-
50 tected in postexercise samples (E), while no
significant correlation could be seen between
0 0 cf-DNA levels and the muscle damage marker
ET UT ET UT ET UT ET UT ET UT ET UT myoglobin (F). mKuU, milli-Kunitz units; GE,
pre post +3h pre post +3h genome equivalents. *P ⬍ 0.05, significant dif-
ference within group. **P ⬍ 0.01, significant
difference within group.
E F
25000 25000
Spearman‘s rho = 0.699, P = 0.011 Spearman‘s rho = -0.028, P = 0.931
cf-DNA (GE/ml plasma)
cf-DNA (GE/ml plasma)

20000 20000

15000 15000

10000 10000

5000 5000

0 0
100 150 200 250 300 20 30 40 50 60 70 80 90
MPO (ng/ml plasma) Myoglobin (ng/ml plasma)

markers myoglobin and FABP; 3) postexercise concentra- erated by exocytosis (43, 52). As indicated by the observed
tions of cf-DNA are significantly correlated with circulating association between plasma levels of cf-DNA and MPO, it
levels of the neutrophil granule enzyme MPO; and 4) has become evident that active release of MPO by neutro-
neutrophils with delobulated nuclei and emanating DNA phils is primarily accomplished via colocalization with the
fiber structures are apparent in postexercise blood smears, chromatin backbone of NETs (55). Consistently, levels of
indicating that intravascular NETs are released in response MPO have been shown to strongly correlate with cf-DNA in
to intense exercise. the blood of patients with thrombotic microangiopathies
In line with our results, degranulation of neutrophils, as (26), and in patients with deep vein thrombosis (17). More-
predicted by an immediate increase of MPO in the circula- over, by use of capture ELISA protocols, true MPO-DNA
tion, has commonly been reported in response to intense complexes have recently been detected in the circulation of
physical exercise (46, 47). Remarkably, the hemoprotein patients with small vessel vasculitis (SVV) (34) and in
MPO is contained within the neutrophil primary granules, individuals with suspected coronary artery disease (6).
which are destined to fuse with the forming or formed NETs were originally described as an innate defense mech-
phagosome after microbial engulfment, but are barely lib- anism to extracellularly capture and potentially kill invading

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330 NETs in Exercise • Beiter T et al.

Table 2. Plasma concentrations of inflammation-associated blood markers at rest, immediately after, and 3 h after 60 min
of intense cycling exercise in endurance-trained and untrained individuals
Pre Post ⫹3 h

ET UT ET UT ET UT

FABP, ng/ml 2.4 1.6 4.1 2.5 4.1 11.1

(1.4–3.4) (1.4–2.4) (2.8–7.3) (1.8–3.4) (3.7–6.9)*,† (2.0–49.4)‡
GH, ng/ml 0.3 0.2 18.8 31.0 0.5 0.2
(0.1–0.7) (0.1–0.3) (6.9–99.0)† (8.4–56.8)† (0.2–1.0) (0.1–0.6)
IL-6, pg/ml 1.1 1.1 4.8 3.7 1.5 2.3
(0.1–2.4) (0.1–5.8) (3.6–11.0)† (1.8–5.7) (1.1–2.6) (1.1–4.0)
IL-8, pg/ml 3.5 4.6 7.3 5.5 5.2 4.1
(2.7–6.9) (3.2–5.9) (5.6–9.9)† (2.7–6.5) (4.9–6.2) (0.1–7.1)
IL-10, pg/ml 3.7 4.2 10.6 5.1 5.0 3.8
(2.8–4.6) (1.9–5.6) (5.3–18.9)‡ (2.7–8.9) (3.9–5.7) (2.1–6.1)
IL-16, pg/ml 190 203 359 291 263 278
(147–306) (151–266) (210–589)‡ (217–435) (185–439) (209–415)
Lactate, mmol/l 1.1 0.9 5.3 5.5
(0.7–1.4) (0.7–1.1) (4.2–8.6)‡ (2.3–7.2)‡
MMP2, ng/ml 2,240 2,070 2,585 2,010 2,325 2,056
(1,840–2,510) (1,620–2,650) (2,324–3,401)† (1,530–2,552) (1,982–2,779) (1,482–2,518)
MMP3, ng/ml 12.5 12.5 22.8 14.8 12.6 9.4
(11.0–17.0) (6.5–17.0) (16.9–27.5)† (9.2–23.6) (10.1–17.33) (5.5–19.1)
MMP9, ng/ml 47 45 80 100 92 140
(19–54) (26–78) (69–101) (55–195)† (45–150)† (44–250)†
MPO, ng/ml 105 122 192 190 118 168

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(88–156) (105–148) (137–232)† (157–274)‡ (74–204) (119–200)
Myoglobin, ng/ml 34 31 60 47 110 327
(28–72) (27–51) (55–77) (26–81) (73–203)*,‡ (50–914)‡
Values are median (range); n ⫽ 6. Pre, at rest; Post, immediately after; ⫹3h, after 60 min of intense cycling exercise; ET, endurance-trained; UT, untrained;
FABP, fatty acid-binding protein in heart; GH, growth hormone; IL, interleukin; MMP, matrix metalloproteinase; MPO, myeloperoxidase. *P ⬍ 0.05, significant
difference compared to UT group at indicated time point. †P ⬍ 0.05, significant difference within group compared to preexercise level. ‡P ⬍ 0.01, significant
difference within group compared to preexercise level.

pathogens (8, 9). In their seminal study, Brinkmann et al. (8)
reported that, on stimulation with PMA, neutrophils undergo a
sequel of cytoplasmic and nuclear changes that culminate in
the rupture of the cellular membrane to release web-like
structures of DNA coated with nuclear and granule proteins
(including MPO). This lytic process of NET release, termed
NETosis, occurs slowly over 2– 4 h, is dependent on formation
of reactive oxygen species, and is clearly distinct from classical
forms of cell death like apoptosis or necrosis. However, despite
extensive research efforts in recent years, the molecular path-
ways and the biological significance of this phenomenon are
just beginning to emerge. It appears that— dependent on stim-
ulus, location, milieu, and activation state—several distinct but
partially overlapping signaling pathways exist that culminate in
the formation of NETs for varying purposes (30, 45). Recently,
Paul Kubes and colleagues (49, 59) uncovered an alternative,
nonlytic and very rapid process of NET formation that does not
require cell death, thereby enabling intravascular neutrophils to
expel their decondensed nuclear contents without losing their
chemotactic and phagocytic abilities. The described phenotype
intriguingly resembles that of the seemingly intact NET-form-
ing neutrophils we observed in the blood smear specimens of
postexercise samples.
There is increasing evidence that release of chromatin serves
as an ancient and highly conserved strategy of host defense (9).
It also appears that NETs evolved to interact at different stages
of innate immune responses (30). In the vasculature, NETs
recently emerged as being crucially important to accomplish
bidirectional crosstalk between innate immunity and blood
coagulation, a task that seems inevitable to protect hosts from
non-self and altered-self, and has therefore been termed im-
munothrombosis (21). NETs have been shown to constitute a
scaffold for platelet and red blood cell adhesion and to con-
centrate effector proteins involved in thrombus formation (7,
25, 56). Moreover, the chromatin backbone of NETs provides
a contact surface to initiate the intrinsic coagulation cascade
(25, 44), and diverse NET components have been shown to
antagonize with natural coagulation inhibitors (1, 39). It is
noteworthy that strenuous or exhaustive exercise is known to
elicit a transient hypercoagulable state characterized by platelet
activation as well as selective stimulation of secondary hemo-
stasis via the intrinsic coagulation pathway (20, 36). The
physiological implications and mechanistic basis of this spe-
cific hemostatic response to exercise have been the subject of
long-standing controversy, but could indeed be conclusively
described as potentially dependent on the exercise-induced
formation of intravascular NETs.
Although there are clearly many facets of NETs that require
further exploration, it is quite undisputed that appropriate
functioning of NETs is an important facet of effective host
defense. On the other hand, aberrant or unresolved release of
NETs can correlate with disease severity, imposes an increased
risk of thrombotic events, may contribute to vascular injury
and tissue damage, and has been implicated in the etiology of
autoimmunity (9, 30). As complexes of self-DNA and neutro-
phil-derived granule proteins provide a source of autoantigens
and have been identified as potent activators of dendritic cells,
NETs just recently emerged as key players in atherogenesis
(18, 19) and in the development of diverse autoimmune dis-
eases including systemic lupus erythematosus (SLE) (27),

J Appl Physiol • doi:10.1152/japplphysiol.00173.2014 • www.jappl.org

 NETs in Exercise
SVV (51), rheumatoid arthritis (RA) (33), inflammatory skin
disease (29), and type I diabetes (16). Moreover, aberrant
release of NETs has been linked to cancer-associated throm-
bosis, tumor growth, and metastasis (13–15). At first sight, it
might appear paradoxical that exercise should trigger an im-
mune mechanism that seems crucially involved in many
chronic diseases and conditions that regular exercise is meant
to protect one from. However, one might speculate that, by
allowing “safe” release of NETs in a noninflammatory or
tolerogenic environment, exercise-induced formation of intra-
vascular NETs might provide an essential and immanent strat-
egy to establish self-tolerance to neutrophil-derived peptides
and self-DNA, thus contributing significantly to the health
benefits of physical activity.
Irrespective of its functional role(s), timely removal of
exercise-induced NETs should be of indispensable importance
to preserve tissue homeostasis, to avoid thrombotic complica-
tions, and to prevent aberrant presentation of self-antigens (10,
31, 38, 42). Here, we demonstrate that the rapid resolution of
exercise-induced cf-DNA could be attributed to a concomitant
and transient rise in serum DNase 1 activity. DNase 1 is the
most important waste-management nuclease in serum with
responsibility for degrading extracellular DNA. Consistently,
DNase 1 has been depicted as the primary degrader of NETs,
and impairment or deficiency in DNase 1 activity has been
directly linked to the development of autoimmune diseases (31,
38). Remarkably, an increased seroprevalence of antinuclear
antibodies, a hallmark of SLE and RA, has also been reported
in athletes suffering from chronic fatigue (50). Thus, it seems
reasonable to assume that aberrant or unresolved release of
cf-DNA/NETs might profoundly contribute to unanticipated
adverse outcomes of acute or prolonged exercise in susceptible
individuals, evoked, for example, by excessive or unaccus-
tomed training loads, high stress levels, or insufficient rest
periods.
Considering the overall health-promoting effect of regular
exercise, we wondered whether sedentary and physically active
individuals in general display distinct cf-DNA/DNase re-
sponses to acute exercise. Our results indicate that the individ-
ual response to exercise is not directly interconnected to the
training status, at least not in healthy young individuals—
which of course does not rule out that exercise intervention
might affect these responses in susceptible individuals. Like-
wise, it will be important to determine the responsiveness in
individuals with known risk factors for lifestyle-related dis-
eases. Remarkably, patients with severe coronary atheroscle-
rosis or calcified coronary arteries have recently been shown to
display increased plasma levels of cf-DNA, and quantification
of circulating MPO-DNA complexes has been demonstrated of
predictive value for future cardiovascular events (6). In this
way, monitoring of cf-DNA/DNase levels or activities in
response to a standardized exercise testing could provide a
valuable tool to identify people who are at increased risk of
developing cardiac ischemia, thrombosis, autoimmunity, air-
way hyperresponsiveness, or chronic fatigue.
In summary, we provide descriptive evidence of exercise-
induced release of NETs. This observation might open a new
avenue to improve our understanding of exercise immunology.
Future work will show whether this phenomenon is rather a
reminiscence of our ancient fight-or-flight response or whether
J Appl Physiol • doi:10.1152/japplphysiol.00173.2014 • www.jappl.org
• Beiter T et al. 331
it represents a fundamental layer of immune response to
exercise.
GRANTS
Part of this study was supported by the Bundesinstitut für Sportwissen-
schaft, 081902/09-13.
DISCLOSURES
No conflicts of interest, financial or otherwise, are declared by the author(s).
AUTHOR CONTRIBUTIONS
Author contributions: T.B., J.M.S., F.C.M., and A.M.N. conception and
design of research; T.B., A.F., and J.H. performed experiments; T.B. and M.S.
analyzed data; T.B., A.F., and A.M.N. interpreted results of experiments; T.B.
prepared figures; T.B. and A.M.N. edited and revised manuscript; T.B., A.F.,
J.H., M.S., J.M.S., F.C.M., and A.M.N. approved final version of manuscript.
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