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Acute Severe Exercise Facilitates Neutrophil Extracellular Trap Formation in Sedentary but Not Active Subjects

SYU, GUAN-DA1,2; CHEN, HSIUN-ING1,2; JEN, CHAUYING J.1,2

Author Information

1Institute of Basic Medical Sciences, National Cheng Kung University, Tainan, TAIWAN; and 2Department of Physiology, College of Medicine, National Cheng Kung University, Tainan, TAIWAN

Address for correspondence: Professor Chauying J. Jen, Department of Physiology, National Cheng Kung University, Tainan 701, Taiwan; E-mail: [email protected].

Submitted for publication June 2012.

Accepted for publication August 2012.

Supplemental digital content is available for this article. Direct URL citations 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).

Medicine & Science in Sports & Exercise: February 2013 - Volume 45 - Issue 2 - p 238-244
doi: 10.1249/MSS.0b013e31826df4a1

Abstract

Neutrophils play a critical role in the first line of defense against pathogens. After reaching the infection 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 proteins, which are capable of trapping and killing pathogens (2,3,11). NET formation is highly dependent on ROS and nicotinamide adenine denucleotide phosphate (NADPH) oxidase. There are two lines of evidence supporting the involvement 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 role of NETs in innate immunity, dysregulated NET formation is often associated with autoimmune diseases and inflammatory 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 under physiological conditions is unknown. Acute severe exercise (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 α, interleukin (IL) 1β, 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 potential (ΔΨm), 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 physically 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 ΔΨm was further investigated.

METHODS

Subjects.

The protocol was reviewed and approved by the Human Ethics Committee of National Cheng Kung 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.

T1-4
TABLE 1:
Basic anthropometric parameters, exercise performance, and leukocyte count in sedentary and active groups.

Exercise paradigms and blood collection.

Subjects in both groups underwent a single bout of ASE. They arrived at 9:00 a.m., rested for approximately 30 min, and performed the ASE on a cycle ergometer (E3200HRT; Vision 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 analyzer (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 balanced salt solution. Contaminating erythrocytes were removed by a 30-s hypotonic shock. Neutrophils were than resuspended at 5 × 106 cells per milliliter in Hank’s balanced salt solution. These neutrophils were either used immediately to determine the cytosolic ROS, glutathione level, and ΔΨm or cultured at 37°C for 3 h to determine the NET formation. The purity and the viability (>95%, immediately after isolation) were routinely checked by Wright’s stain and trypan blue exclusion, respectively.

Determination of the redox status and ΔΨm 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 ΔΨm was quantified by measuring the red fluorescence intensity of JC-1 (35). Freshly isolated neutrophils were stained with DCF-DA (1 μM; Sigma-Aldrich), mBBr (50 μM; Sigma-Aldrich), or JC-1 (7.7 μM; 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.

Determination of NET formation.

Because a microplate reader detected both extracellular DNA from NETs forming cells and intracellular DNA from membrane disrupted/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 μM; 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 μM; Sigma-Aldrich), and cyanide-p-trifluoromethoxyphenylhydrazone (FCCP, 100 nM; Sigma-Aldrich) were used to remove ROS, to inhibit NADPH oxidase, and to suppress ΔΨm, respectively. These inhibitors were individually added and incubated for another 20 min. Neutrophils were then incubated in the presence or absence of PMA (100 nM) at 37°C for 3 h before microscopy analyses. Neutrophil images from five fixed regions 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 calculated as (NETs forming cell count/total cell count) × 100%.

Statistical analysis.

Two-way repeated-measures ANOVA followed by Bonferroni posttest was used to analyze the ASE effects and group differences or ASE effects and inhibitors effects. Significant differences were defined as P < 0.05. All data were presented as mean ± SEM, where n is the number of subjects.

RESULTS

Basic anthropometric parameters, exercise performance, and leukocyte count in sedentary and active groups.

Results in Table 1 show the basic anthropometric parameters, exercise performance, and leukocyte count in sedentary and active groups. Anthropometric 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 sedentary controls. The leukocyte count increased immediately after 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 when labeled with Sytox Orange. Compiled fluorescent micrographs 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.

F1-4
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.
F2-4
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 < 0.05, post-ASE vs pre-ASE; # P < 0.05, active vs sedentary; n = 10.

Effects of ASE on neutrophil basal redox status and ΔΨm.

We further investigated the possible involvement of redox status and mitochondria function in ASE-induced NET formation. Cytosolic ROS, glutathione level, and ΔΨm were determined in freshly isolated neutrophil (Fig. 3). At the resting state, the active group showed higher glutathione level, whereas both groups showed similar cytosolic ROS level and ΔΨm. A single bout of ASE in the sedentary group increased neutrophil cytosolic ROS, decreased glutathione level, and suppressed ΔΨm. As a comparison, ASE in the active group did not alter the redox status and ΔΨm. Taken together, ASE in sedentary but not active subjects suppressed ΔΨm and augmented oxidative stress in neutrophils.

F3-4
FIGURE 3:
Effects of ASE on neutrophil basal redox status and ΔΨm. The cytosolic ROS, glutathione level, and ΔΨm were examined in the freshly isolated neutrophils. Data were analyzed by two-way repeated-measures ANOVA followed by Bonferroni posttest. *P < 0.05, post-ASE vs pre-ASE; #P < 0.05, active vs sedentary; n = 10.

The role of ROS in ASE-facilitated NET formation.

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.

F4-4
FIGURE 4:
Effects of ROS and ΔΨm 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 ΔΨm, 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 < 0.05, post-ASE vs pre-ASE; §P < 0.05, treated vs untreated; n = 10.

The role of ΔΨm in ASE-facilitated NET formation.

Our results showed that ASE in sedentary but not active groups suppressed neutrophil ΔΨm and facilitated NET formation. Whether the suppressed ΔΨm also plays a role in ASE-facilitated NET formation deserves further investigation. Indeed, the NET formation was enhanced by adding FCCP to suppress ΔΨm (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 ΔΨm 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 oxidative stress and reduced ΔΨm, which facilitated NET formation in both basal and PMA-stimulated conditions. In contrast, ASE in active subjects did not facilitate NET formation and showed no such effects on neutrophil oxidative stress and ΔΨm. 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 incapable of neutralizing the excessive oxidative stress generated 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 production are likely due to not only differences in physical fitness of subjects but also differences in laboratory assay techniques (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 former technique is sensitive to intracellular hypochlorous acid, whereas the latter technique is sensitive to extracellular superoxide instead (5,22). In this study, we used the DCF-DA method because of its specificity in detecting cytosolic hydrogen 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 mitochondria, or the neutrophil NADPH oxidase. ROS released from skeletal muscles under ASE transiently tips the redox balance in the blood stream toward pro-oxidative state (9,32), which would elevate the cytosolic ROS in neutrophils. 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 experiments in this study indicated that the ASE-facilitated NET formation depended on NADPH oxidase-generated ROS (Fig. 4). Besides, overtraining in rats activates neutrophil 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 because 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 determinant 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 instead (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 neutrophils (2,31), whereas apoptosis is usually assayed using suspended neutrophils (10,35). Nevertheless, exogenous addition of 100 μM 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 sedentary 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 ΔΨm 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 ΔΨm during ASE challenge (36). Therefore, the decrease in ΔΨm 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 ΔΨm regulates NET formation is unclear at present. In our opinion, a suppressed ΔΨm somehow activated NADPH oxidase as the FCCP-facilitated NET formation diminished in the presence of NAC or DPI (see Figure, Supplemental Digital Content 1, https://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 together, both ΔΨm 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 (this study) and spontaneous apoptosis in sedentary subjects (35). Therefore, neutrophils are likely to be one of the factors that responsible for the weakened immunity after excessive 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 unnecessary tissue damage and inflammation. As a whole, ASE in sedentary subjects exerts adverse effects on neutrophils 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 altering 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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Keywords:

MITOCHONDRIA; NADPH OXIDASE; NEUTROPHIL; NETOSIS; PHYSICAL FITNESS; ROS

©2013The American College of Sports Medicine