Acute Severe Exercise Facilitates Neutrophil

Extracellular Trap Formation in Sedentary but

Not Active Subjects
GUAN-DA SYU1,2, HSIUN-ING CHEN1,2, and CHAUYING J. JEN1,2
1
Institute of Basic Medical Sciences, National Cheng Kung University, Tainan, TAIWAN; and 2Department of Physiology,
College of Medicine, National Cheng Kung University, Tainan, TAIWAN

ABSTRACT
SYU, G.-D., H.-I. CHEN, and C. J. JEN. Acute Severe Exercise Facilitates Neutrophil Extracellular Trap Formation in Sedentary but Not
Active Subjects. Med. Sci. Sports Exerc., Vol. 45, No. 2, pp. 238–244, 2013. Neutrophil extracellular trap (NET), a newly revealed
antimicrobial strategy, is usually evoked by reactive oxygen species (ROS) and nicotinamide adenine denucleotide phosphate (NADPH)
BASIC SCIENCES

oxidase activation. In addition, the acute severe exercise (ASE)–induced oxidative stress in neutrophils depends on the subject’s physical
fitness. Purpose: We investigated whether ASE exerted differential effects on NET formation in sedentary and physically active subjects.
Methods: Young males, 10 sedentary and 10 physically active, underwent an ASE (pedaling on a bicycle ergometer with increasing
loads until exhaustion). Neutrophils were isolated from blood specimens drawn before and immediately after ASE for assaying NET
formation along with redox-related parameters and mitochondrial membrane potential ($#m). Results: In the sedentary group, 1) after
ASE, NET formation increased spontaneously and in response to stimulation with phorbol 12-myristate 13-acetate; 2) ASE increased
cytosolic ROS, decreased glutathione, and suppressed $#m in neutrophils; 3) removing ROS or inhibiting NADPH oxidase prevented
the ASE-facilitated NET formation; and 4) suppressing $#m prevented the ASE-facilitated NET formation. On the contrary, these ASE
effects on neutrophils did not happen in the active group. Conclusions: ASE in sedentary but not active subjects facilitated NET
formation via elevating the NADPH oxidase-generated ROS and suppressing the $#m. Key Words: MITOCHONDRIA, NADPH
OXIDASE, NEUTROPHIL, NETOSIS, PHYSICAL FITNESS, ROS

N
eutrophils play a critical role in the first line of de-
fense against pathogens. After reaching the infec-
tion site, they ingest pathogens, release reactive
oxygen species (ROS), and even release their own DNA to
form neutrophil extracellular traps (NETs) (2). NETs consist
of DNA, histone, granule proteins, and some cytosolic pro-
teins, which are capable of trapping and killing pathogens
(2,3,11). NET formation is highly dependent on ROS and
nicotinamide adenine denucleotide phosphate (NADPH) oxi-
dase. There are two lines of evidence supporting the involve-
ment of ROS and NADPH oxidase in NET formation. First,
NET formation is evoked by exogenous oxidants and inhibited
by antioxidants (11). Second, neutrophils from patients with
impaired NADPH oxidase fail to form NETs (1). Despite the
Address for correspondence: Professor Chauying J. Jen, Department of
Physiology, National Cheng Kung University, Tainan 701, Taiwan; E-mail:
jen@mail.ncku.edu.tw.
Submitted for publication June 2012.
Accepted for publication August 2012.
Supplemental digital content is available for this article. Direct URL cita-
tions appear in the printed text and are provided in the HTML and PDF
versions of this article on the journal_s Web site (www.acsm-msse.org).
0195-9131/13/4502-0238/0
MEDICINE & SCIENCE IN SPORTS & EXERCISEÒ
Copyright Ó 2012 by the American College of Sports Medicine
DOI: 10.1249/MSS.0b013e31826df4a1
role of NETs in innate immunity, dysregulated NET formation
is often associated with autoimmune diseases and inflamma-
tory diseases, such as lupus erythematosis (14,18), cystic
fibrosis (20), gout (21), preeclampsia (13), and sepsis (6).
How NET formation in healthy subjects is regulated un-
der physiological conditions is unknown. Acute severe ex-
ercise (ASE) increases oxidative stress, induces damages in
the skeletal muscles (25,26), and augments both pro- and
anti-inflammatory cytokines (e.g., tumor necrosis factor >,
interleukin (IL) 1A, IL-1 receptor antagonist, IL-6, and IL-10)
(29,33). However, the ASE responses are dependent on the
subject’s physical fitness. Our recent studies show that ASE
augments ROS, suppresses mitochondrial membrane poten-
tial ($#m), and accelerates apoptosis in neutrophils isolated
from sedentary subjects but not from the exercise trained
subjects (35,36). We thus hypothesized that ASE exerted
differential effects on NET formation in sedentary and phys-
ically active subjects. In this study, we aimed to characterize
the ASE effects on NET formation in sedentary and active
subjects. In addition, the possible involvement of ASE-
evoked changes in ROS and $#m was further investigated.
METHODS
Subjects. The protocol was reviewed and approved by
the Human Ethics Committee of National Cheng Kung

238

Copyright © 2013 by the American College of Sports Medicine. Unauthorized reproduction of this article is prohibited.
University Medical College (Institutional Review Board No.
ER-96-92). Written informed consent was received from all
participants. The exercise paradigms were modified from
our previous reports (35,37). Young males, 10 sedentary and
10 active, participated in this study. They all fulfilled the
general requirements: no smoking, no previous record of
metabolic or cardiovascular diseases, no recent symptoms of
upper respiratory tract infection, and abstained from any
medication for at least 1 month before the study. Besides
general requirements, subjects in the sedentary group were
not involved in regular exercise (less than once per week) in
the past 6 months whereas subjects in the active group were
involved in regular exercise (more than three times per week)
in the past 6 months. For the basic anthropometric parameters,
see Table 1.
Exercise paradigms and blood collection. Subjects
in both groups underwent a single bout of ASE. They ar-
rived at 9:00 a.m., rested for approximately 30 min, and
performed the ASE on a cycle ergometer (E3200HRT; Vi-
sion Fitness, Madison, WI) with continuous increments of
workload every 3 min until exhaustion. The heart rate
reached at least 90% of the predicted maximal heart rate at
the end of ASE. Peripheral venous blood samples were
drawn at rest before (pre-ASE) and immediately after ASE
(post-ASE). Blood was collected into vacutainers containing
sodium citrate and then stored on ice. The leukocyte and
granulocyte count were measured using a hematology ana-
lyzer (KX-21N; Sysmex, Mundelein, IL).
Neutrophil isolation and culture. Neutrophils were
purified by histopaque density gradient (10771 and 11191;
Sigma-Aldrich, St. Louis, MO) and washed in Hank’s bal-
anced salt solution. Contaminating erythrocytes were re-
moved by a 30-s hypotonic shock. Neutrophils were than
resuspended at 5  106 cells per milliliter in Hank’s bal-
anced salt solution. These neutrophils were either used im-
mediately to determine the cytosolic ROS, glutathione level,
and $#m or cultured at 37-C for 3 h to determine the NET
formation. The purity and the viability (995%, immediately
after isolation) were routinely checked by Wright’s stain and
trypan blue exclusion, respectively.
TABLE 1. Basic anthropometric parameters, exercise performance, and leukocyte count
in sedentary and active groups.
Sedentary (n = 10)
Age (yr) 23 T 0
Height (cm) 174 T 2
Weight (kg) 66 T 2
BMI (kgImj2) 21.7 T 0.5
Resting heart rate (bpm) 75 T 2
Maximum heart rate (bpm) 191 T 2
ASE duration (min) 34 T 1
Maximum workload (W) 115 T 4
Leukocyte count pre-ASE (103 KLj1) 5.2 T 0.3
Leukocyte count post-ASE (103 KLj1) 8.8 T 0.4*
Granulocyte count pre-ASE (103 KLj1) 3.4 T 0.2
Granulocyte count post-ASE (103 KLj1) 4.9 T 0.3*
Data were analyzed by two-way repeated-measures ANOVA followed by Bonferroni
posttest. n = 10.
* P G 0.05, post-ASE vs pre-ASE.
† P G 0.05, active vs sedentary.
EXERCISE AND NEUTROPHIL EXTRACELLULAR TRAPS
Copyright © 2013 by the American College of Sports Medicine. Unauthorized reproduction of this article is prohibited.
Determination of the redox status and $#m in
freshly isolated neutrophils. Neutrophil cytosolic ROS
was estimated by DCF-DA fluorescence (35). Intracellular
glutathione level was indicated by monobromobimane
(mBBr) fluorescence (15). Neutrophil $#m was quantified
by measuring the red fluorescence intensity of JC-1 (35).
Freshly isolated neutrophils were stained with DCF-DA
(1 KM; Sigma-Aldrich), mBBr (50 KM; Sigma-Aldrich),
or JC-1 (7.7 KM; Invitrogen, Carlsbad, CA) at 37-C for
30 min. Fluorescent stained cells were then loaded to the
96-well optical-bottom plate (165305; Thermo Scientific,
Rochester, NY) and allowed to attach for 30 min. The
fluorescence intensity was measured by microplate reader
(Synergy HT; BioTek, Winooski, VT) before and after
adding phorbol 12-myristate 13-acetate (PMA; 100 nM,
10 min). Note that because mBBr is light sensitive, all
procedures were executed away from light.
BASIC SCIENCES
Determination of NET formation. Because a micro-
plate reader detected both extracellular DNA from NETs
forming cells and intracellular DNA from membrane dis-
rupted/apoptotic cells, we chose the microscopy method for
NET quantification (4,28). Freshly isolated neutrophils were
allowed to attach to the cell culture plate for 30 min in the
presence of cell-permeable (Syto Green, 1 KM; Invitrogen)
and cell-impermeable (Sytox Orange, 500 nM; Invitrogen)
nucleic acid stains. N-acetyl-L-cysteine (NAC, 5 mM; Sigma-
Aldrich), diphenyleneiodonium chloride (DPI, 10 KM; Sigma-
Aldrich), and cyanide-p-trifluoromethoxyphenylhydrazone
(FCCP, 100 nM; Sigma-Aldrich) were used to remove ROS, to
inhibit NADPH oxidase, and to suppress $#m, respectively.
These inhibitors were individually added and incubated for
another 20 min. Neutrophils were then incubated in the pres-
ence or absence of PMA (100 nM) at 37-C for 3 h before
microscopy analyses. Neutrophil images from five fixed re-
gions were acquired to cover approximately 60% of the total
area. Cell count and NET count in micrographs were quantified
using commercial software (Image ProPlus, Media Cybernetics,
Bethesda, MD). More than 500 cells were analyzed in each
sample. The total number of cells was determined by Syto Green
fluorescence. The NET-forming cell was defined as a Sytox
Orange fluorescence subject larger than a normal neutrophil
image (4,28). The percentage of NET formation was calcu-
lated as (NETs forming cell count/total cell count)  100%.
Active (n = 10) Statistical analysis. Two-way repeated-measures
22 T 1 ANOVA followed by Bonferroni posttest was used to an-
172 T 2 alyze the ASE effects and group differences or ASE effects
65 T 2
21.9 T 0.6 and inhibitors effects. Significant differences were defined
64 T 4† as P G 0.05. All data were presented as mean T SEM,
188 T 2
50 T 2†
where n is the number of subjects.
169 T 6†
5.5 T 0.4 RESULTS
8.7 T 0.6*
3.6 T 0.4 Basic anthropometric parameters, exercise per-
5.0 T 0.6*
formance, and leukocyte count in sedentary and
active groups. Results in Table 1 show the basic anthro-
pometric parameters, exercise performance, and leuko-
cyte count in sedentary and active groups. Anthropometric
Medicine & Science in Sports & Exercised 239
 parameters were the same between groups except that active
subjects showed lower resting heart rate than sedentary subjects.
Subjects in the active group had better exercise performance
(longer ASE duration and higher maximal workload) than sed-
entary controls. The leukocyte count increased immediately af-
ter ASE (the ASE-evoked leukocytosis) in both sedentary and
active groups.
Effects of ASE on NET formation. Neutrophils were
isolated before and immediately after ASE. These cells were
labeled with fluorescent DNA dyes and cultured for 3 h with or
without PMA. The nuclei of viable neutrophils were visible as
small spots when labeled with Syto Green, a cell-permeable
nucleic acid stain. However, they were invisible when labeled
with Sytox Orange, a cell-impermeable nucleic acid stain. As a
comparison, NET-forming neutrophils showed diffused images
BASIC SCIENCES
when labeled with Sytox Orange. Compiled fluorescent micro-
graphs showed the NET formation in sedentary and active
groups (Fig. 1). Quantitative data are summarized in Figure 2.
At the resting state, the basal NET formation was minimal, and
it increased greatly in the presence of PMA. Moreover, there
were no between-group differences in either basal or PMA-
stimulated conditions. In the sedentary group, both basal and
PMA-stimulated NET formation elevated after a single bout of
ASE. In contrast, similar ASE effects were absent in the active
group. Taken together, NET formation was facilitated by ASE
in sedentary but not active groups.
Effects of ASE on neutrophil basal redox status
and $#m. We further investigated the possible involve-
ment of redox status and mitochondria function in ASE-
induced NET formation. Cytosolic ROS, glutathione level,

FIGURE 1—Microscopic observation of NET formation. Neutrophils from sedentary or active groups were isolated before and immediately after
ASE. These neutrophils were incubated in the absence (A) or presence (B) of PMA for 3 h to determine the morphological changes of the nucleus. Syto
Green is a cell-permeable nucleic acid stain, and Sytox Orange is a cell-impermeable nucleic acid stain.

240 Official Journal of the American College of Sports Medicine http://www.acsm-msse.org

Copyright © 2013 by the American College of Sports Medicine. Unauthorized reproduction of this article is prohibited.
FIGURE 2—Effects of ASE on NET formation in sedentary and physically active groups. The NET formation in the absence (A) or presence (B) of
PMA was determined by microscopic counting. The percentage of NET formation was calculated as (NETs forming cell count / total cell count) 
100%. Data were analyzed by two-way repeated-measures ANOVA followed by Bonferroni posttest. *P G 0.05, post-ASE vs pre-ASE; #P G 0.05, active
vs sedentary; n = 10.
and $#m were determined in freshly isolated neutrophil
(Fig. 3). At the resting state, the active group showed higher
glutathione level, whereas both groups showed similar cy-
tosolic ROS level and $#m. A single bout of ASE in the
sedentary group increased neutrophil cytosolic ROS, de-
creased glutathione level, and suppressed $#m. As a com-
parison, ASE in the active group did not alter the redox
status and $#m. Taken together, ASE in sedentary but not
active subjects suppressed $#m and augmented oxidative
stress in neutrophils.
The role of ROS in ASE-facilitated NET forma-
tion. Oxidative stress is a well-known factor in NET
formation. To investigate its role in ASE-facilitated NET
formation, we removed ROS from cultured neutrophils by
adding either NAC (an antioxidant) or DPI (a NADPH
oxidase inhibitor). The NET formation was effectively
blocked by either NAC or DPI (Fig. 4). Moreover, in
the sedentary group, these reagents also blocked the ASE-
facilitated NET formation in vitro.
The role of $#m in ASE-facilitated NET forma-
tion. Our results showed that ASE in sedentary but not ac-
tive groups suppressed neutrophil $#m and facilitated NET
formation. Whether the suppressed $#m also plays a role in
ASE-facilitated NET formation deserves further investiga-
tion. Indeed, the NET formation was enhanced by adding
FCCP to suppress $#m (Fig. 4). Moreover, in the sedentary
group, FCCP abolished the ASE-facilitated NET formation
in vitro. Taken together, ASE facilitated NET formation
likely by suppressing the $#m and augmenting the NADPH
oxidase-generated ROS.
DISCUSSION
This study is the first to show that NET formation was
facilitated by ASE in sedentary but not active subjects.
Moreover, ASE in sedentary subjects increased neutrophil
EXERCISE AND NEUTROPHIL EXTRACELLULAR TRAPS
Copyright © 2013 by the American College of Sports Medicine. Unauthorized reproduction of this article is prohibited.
BASIC SCIENCES
oxidative stress and reduced $#m, which facilitated NET
formation in both basal and PMA-stimulated conditions. In
contrast, ASE in active subjects did not facilitate NET for-
mation and showed no such effects on neutrophil oxidative
stress and $#m. Although the release of NETs is important
in antimicrobial function, NET formation in the absence of
microbial infections can be harmful to the host, such as
causing tissue damages or autoimmune disorders (19,31).
Our results indicated that ASE in sedentary subjects made
some neutrophils hyperactive and thus disturbed the immune
regulation as a whole.
Oxidative stress is one of the most important factors to
induce NET formation (11). As neutrophils from sedentary
subjects had low glutathione content (Fig. 3), they were in-
capable of neutralizing the excessive oxidative stress gen-
erated from ASE. Therefore, the ASE-facilitated NET
formation happened in sedentary subjects but not in active
subjects (Fig. 4). Some disparate findings in literature about
whether and how exercise evokes neutrophil ROS produc-
tion are likely due to not only differences in physical fitness
of subjects but also differences in laboratory assay tech-
niques (30,34). The exercise-induced changes in neutrophil
ROS production depend on the techniques used, that is,
the ASE-evoked ROS production in neutrophils is detected
by using luminol-enhanced chemiluminescence but not by
using lucigenin-enhanced chemiluminescence (34). The for-
mer technique is sensitive to intracellular hypochlorous acid,
whereas the latter technique is sensitive to extracellular su-
peroxide instead (5,22). In this study, we used the DCF-DA
method because of its specificity in detecting cytosolic hydro-
gen peroxide. Our findings are consistent with results obtained
from using the luminol-enhanced chemiluminescence (34).
The ASE-induced oxidative stress in neutrophils could
have come from the blood-borne ROS, the neutrophil mito-
chondria, or the neutrophil NADPH oxidase. ROS released
from skeletal muscles under ASE transiently tips the redox
Medicine & Science in Sports & Exercised 241

BASIC SCIENCES
FIGURE 3—– Effects of ASE on neutrophil basal redox status and
$#m. The cytosolic ROS, glutathione level, and $#m were examined
in the freshly isolated neutrophils. Data were analyzed by two-way
repeated-measures ANOVA followed by Bonferroni posttest. *P G 0.05,
post-ASE vs pre-ASE; #P G 0.05, active vs sedentary; n = 10.
balance in the blood stream toward pro-oxidative state
(9,32), which would elevate the cytosolic ROS in neu-
trophils. As neutrophils were washed several times during
the isolation procedure, the blood-borne ROS should exert
minimal effects on neutrophil functions in vitro. In principle,
mitochondrial ROS could serve as an endogenous source for
ASE-induced oxidative stress in neutrophils. However, this
possibility is low because the mitochondrial ROS in post-
ASE neutrophils remains at basal levels for approximately
10 h after isolation (35). In contrast, results from inhibitor
242 Official Journal of the American College of Sports Medicine
Copyright © 2013 by the American College of Sports Medicine. Unauthorized reproduction of this article is prohibited.
experiments in this study indicated that the ASE-facilitated
NET formation depended on NADPH oxidase-generated
ROS (Fig. 4). Besides, overtraining in rats activates neutro-
phil NADPH oxidase (8). Therefore, ROS from neutrophil
NADPH oxidase is likely the key factor responsible for ASE-
facilitated NET formation in sedentary subjects.
Our recent study demonstrates that ASE in sedentary
subjects accelerates neutrophil spontaneous apoptosis be-
cause of elevated oxidative stress (35). Interestingly, these
ASE effects diminish after these subjects have gone through
a moderate-intensity exercise training for 2 months. ASE
apparently exerts parallel effects on NET formation and
neutrophil apoptosis. Although ROS is also a major deter-
minant for neutrophil apoptosis (12), there are fundamental
differences between NET formation and apoptosis. First, the
apoptotic nucleus exhibits strongly condensed chromatin,
whereas the NET nucleus shows expanded chromatin in-
stead (11,31). Second, the nuclear condensation step in
neutrophil apoptosis takes approximately 10 h, whereas
NET formation completes with releasing of DNA in 3 h
(31,35). Third, NET formation happens in attached neu-
trophils (2,31), whereas apoptosis is usually assayed using
suspended neutrophils (10,35). Nevertheless, exogenous
addition of 100 KM hydrogen peroxide mimicked the ASE
effects on oxidative stress and apoptosis (35). It is worth to
note that the same treatment also induces NET formation
(23). Therefore, the ASE-evoked oxidative stress in seden-
tary subjects should be responsible for facilitating both NET
formation and neutrophil apoptosis. As a comparison, ASE
in active subject did not evoke sufficient ROS to facilitate
either NET formation or neutrophil apoptosis.
Results from our FCCP experiments indicated that $#m
reduction is another factor favoring NET formation. As the
neutrophil citrate synthase activity in sedentary subjects is
lower than that in trained subjects, neutrophils in sedentary
subjects are unable to sustain the $#m during ASE chal-
lenge (36). Therefore, the decrease in $#m occurred after
ASE in sedentary subjects but not in active subjects (Fig. 3).
Recently, mitochondrial DNA has been identified in the
NET structure (16), which indicates a possible regulating
role of mitochondria during NET formation. Exactly how
$#m regulates NET formation is unclear at present. In our
opinion, a suppressed $#m somehow activated NADPH
oxidase as the FCCP-facilitated NET formation diminished
in the presence of NAC or DPI (see Figure, Supplemental
Digital Content 1, http://links.lww.com/MSS/A188; which
demonstrates the mechanisms of FCCP-facilitated NET
formation). A possible mechanism may involve the release
of calcium ions from malfunctioning mitochondria and
subsequent activation of NADPH oxidase (7). Taken to-
gether, both $#m reduction and oxidative stress contributed
to the ASE-facilitated NET formation in sedentary subjects.
Excessive exercise produces an open window for 3–72 h,
during which exercising individuals are more susceptible to
infection (17). Here we demonstrated that ASE shortened
neutrophil lifespan via accelerating both NET formation
http://www.acsm-msse.org
 BASIC SCIENCES
FIGURE 4—Effects of ROS and $#m on ASE-facilitated NET formation. Several inhibitors were used to investigate the role of oxidative stress and
depolarized mitochondria on ASE-facilitated NET formation. Neutrophils were pretreated for 30 min with NAC, DPI, or FCCP to remove ROS, to
inhibit NADPH oxidase, or to suppress $#m, respectively. These neutrophils were further incubated in the absence (A and B) or presence (C and D)
of PMA for 3 h to determine the NET formation. Data were analyzed by two-way repeated-measures ANOVA followed by Bonferroni posttest.
*P G 0.05, post-ASE vs pre-ASE; §P G 0.05, treated vs untreated; n = 10.

(this study) and spontaneous apoptosis in sedentary subjects
(35). Therefore, neutrophils are likely to be one of the fac-
tors that responsible for the weakened immunity after ex-
cessive exercise. Besides neutrophil lifespan, ASE also
changes many neutrophil functions in the sedentary subjects,
for example, increases chemotaxis and oxidative burst
(35,36). These hyperactive neutrophils may produce un-
necessary tissue damage and inflammation. As a whole,
ASE in sedentary subjects exerts adverse effects on neu-
trophils and thus disturbs normal immunity.
Regular exercise in humans generally improves immunity,
for example, it lowers the susceptibility to viral and bacterial
infections (24,38). Neutrophils play essential roles in the host
defense against bacteria. Their major antibacterial strategies
include phagocytosis, degranulation, oxidative burst, and
NET formation. Phagocytosis is a very rapid process that
causes minimal damages to host cells (27). In comparison,
degranulation, oxidative burst, and NET formation take a
longer time to activate and damage both pathogens and host
cells. Regular exercise improves phagocytosis (36) without al-
tering oxidative burst (35) or NET formation (this study). Thus,
neutrophil functions improve when sedentary subjects exercise
regularly. Taken together, our present and previous studies
shed light on cellular mechanisms explaining the complex
interactions between different physical activities and neutrophil
functional performance.
This study was supported by the National Science Council, Taiwan
(grant nos. NSC 96-2320- B-006-003, NSC 98-2320-B-006-019-MY3,
and NSC 98-2320-B-006-028-MY3).
The authors thank Ms M. F. Chen for blood drawing, and Ms S. Y.
Hu for subject and exercise arrangements.
The authors have no conflict of interest to declare.
The results of the present study do not constitute endorsement by
the American College of Sports Medicine.

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