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Impact of Physical Activity on Monocyte Subset CCR2 Expression and
Macrophage Polarization Following Moderate Intensity Exercise
Article · December 2019
DOI: 10.1016/j.bbih.2019.100033
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Full Length Article
Impact of physical activity on monocyte subset CCR2 expression and
macrophage polarization following moderate intensity exercise
Anson M. Blanks, Thomas T. Wagamon, Lindsay Lafratta, Mabel G. Sisk, Morgan B. Senter,
Lauren N. Pedersen, Natalie Bohmke, Attiya Shah, Virginia L. Mihalick, R. Lee Franco
*
Department of Kinesiology and Health Sciences, College of Humanities and Sciences, Virginia Commonwealth University, Richmond, VA, United States
ARTICLE INFO
Keywords:
Monocytes
Macrophages
CCR2
Physical activity
Exercise
ABSTRACT
Coronary artery disease (CAD) is an immune-mediated disease in which CCR2 attracts classical, intermediate, and
non-classical monocytes to the arterial intima where they differentiate to macrophages. Balance between pro-
inflammatory M1 and anti-inflammatory M2 macrophages contributes to CAD prevention. Moderate to
vigorous intensity physical activity (MVPA) elicits an immune response and reduces the incidence of CAD,
however, the impact of prior MVPA on monocyte subset CCR2 expression and macrophage polarization following
acute exercise is unknown.
Purpose: To determine the impact of physical activity status on monocyte subset CCR2 surface expression and
macrophage polarization in response to an acute bout of moderate intensity cycle ergometry.
Methods: 24 healthy women and men (12 high physically active [HIACT]: 1500 METmin/wk MVPA &12 low
physically active [LOACT]: <600 METmin/wk MVPA) underwent an acute moderate intensity (60% VO
2peak
)
bout of cycle ergometry for 30 min. Blood samples were collected prior to (PRE), immediately (POST), 1 h (1H),
and 2 h (2H) following exercise. Monocyte CCR2 and macrophage CD86 (M1) and CD206 (M2) were analyzed by
flow cytometry.
Results: Intermediate monocyte CCR2 decreased in response to exercise in the HIACT group (PRE: 11409.0
1084.0 vs. POST: 9524.3 1062.4; p ¼0.034). Macrophage CD206 was lower in the LOACT compared to the
HIACT group at 1H (HIACT: 67.2 5.6 vs. LOACT: 50.1 5.2%; p ¼0.040). Macrophage CD206 at 1H was
associated with both PRE (r ¼0.446, p ¼0.043) and POST (r ¼0.464, p ¼0.034) non-classical monocyte CCR2.
Conclusion: These data suggest that regular moderate to vigorous physical activity positively impacts both
monocytes and macrophages following acute moderate intensity exercise and that this impact may contribute to
the prevention of coronary artery disease.
1. Introduction
Coronary artery disease (CAD), the most common form of cardio-
vascular disease, is the leading cause of death in developed nations
(Mozaffarian et al., 2015). Although preventative pharmacological in-
terventions have been shown to reduce the incidence of CAD, to date, the
most effective prevention strategy is habitual physical activity (Agarwal,
2012).
CAD is a pro-inflammatory immune-mediated disease in which che-
mokines and chemokine receptors, such as C–C chemokine ligand 2
(CCL2, also known as MCP-1) and C–C chemokine receptor (CCR2), are
critical for the attraction of various leukocytes to the arterial intima
(Moore and Tabas, 2011). During an acute pro-inflammatory immune
response, such as following antigen activation or tissue damage, one of
the first and most highly recruited cell types are monocytes (Moore and
Tabas, 2011). Monocytes are divided into three phenotypically and
functionally distinct subsets based on surface expression of CD14 and
CD16 receptors (Mukherjee et al., 2015). Under homeostatic conditions,
classical monocytes (CD14
þþ
CD16
) are released daily from bone
marrow (Patel et al., 2017) and are anti-inflammatory due to the high
level production of the hallmark anti-inflammatory cytokine IL-10 (Wong
et al., 2011). The intermediate (CD14
þþ
CD16
þ
) and non-classical
(CD14
Low
CD16
þþ
) subsets are considered to be more mature
pro-inflammatory monocytes due to the production of cytokines IL-1β
* Corresponding author. Department of Kinesiology and Health Sciences, College of Humanities and Sciences, Virginia Commonwealth University, 1020 West Grace
Street, Room 111, Richmond, VA, 23284.
E-mail address: francorl@vcu.edu (R.L. Franco).
Contents lists available at ScienceDirect
Brain, Behavior, &Immunity - Health
journal homepage: www.editorialmanager.com/bbih/default.aspx
https://doi.org/10.1016/j.bbih.2019.100033
Received 2 December 2019; Received in revised form 20 December 2019; Accepted 21 December 2019
Available online xxxx
2666-3546/©2019 Published by Elsevier Inc. This is an open access article under the CC BY-NC-ND license (http://creativecommons.org/licenses/by-nc-nd/4.0/).
Brain, Behavior, &Immunity - Health xxx (xxxx) xxx
Please cite this article as: Blanks, A.M. et al., Impact of physical activity on monocyte subset CCR2 expression and macrophage polarization following
moderate intensity exercise, Brain, Behavior, &Immunity - Health , https://doi.org/10.1016/j.bbih.2019.100033
and TNF-
α
(Mukherjee et al., 2015;Ong et al., 2018). In addition, in-
termediate and non-classical monocytes are responsible for the ingestion
of debris, as well as extra-cellular matrix breakdown necessary for tissue
repair (Mukherjee et al., 2015;Wong et al., 2011;Ong et al., 2018). Once
within the tissue, monocytes differentiate into macrophages, which are
broadly classified into pro-inflammatory M1 or anti-inflammatory M2
phenotypes (Moore and Tabas, 2011;Italiani and Boraschi, 2014). M1
phenotypes are elicited in response to pro-inflammatory antigen activa-
tion and/or a pro-inflammatory cellular microenvironment and this
macrophage phenotype is responsible for pathogen destruction via the
oxidative burst, as well as extracellular matrix breakdown following
damage (Italiani and Boraschi, 2014;Gonzalez-Dominguez et al., 2016).
In the absence of pro-inflammatory activation or in presence of an
anti-inflammatory microenvironment, macrophages develop an
anti-inflammatory M2 phenotype, which is responsible for immuno-
surveillance and collagen deposition (Italiani and Boraschi, 2014;Boy-
ette et al., 2017). Under normal regulation, proper M1/M2 macrophage
balance contributes to CAD prevention (Moore et al., 2013). However, an
increase in the M1 phenotype leads to greater production of
pro-inflammatory cytokines, eliciting a microenvironment that switches
the M2 phenotype toward the M1 phenotype, which results in the
skewing of macrophage balance toward the M1 phenotype (Moore et al.,
2013). This process precipitates sustained low-grade inflammation and
accumulation of lipid rich macrophages, which eventually progress into
foam cells and atherosclerotic lesions (Moore and Tabas, 2011).
Moderate to vigorous intensity physical activity (MVPA) is known to
elicit a transient immune response, which includes monocytosis (Shinkai
et al., 1992). This response increases monocyte turnover (Claycombe
et al., 2008), increasing anti-inflammatory monocytes in circulation
(Timmerman et al., 2008). These anti-inflammatory monocytes promote
an anti-inflammatory microenvironment which contributes to the
maintenance of M1/M2 macrophage balance in tissue, thereby reducing
the incidence of CAD (Moore et al., 2013;Wattananit et al., 2016).
Regardless of activity status, acute MVPA induces an immune response
and repeated bouts of activity have been shown to alter leukocyte
phenotype and function (Ruffino et al., 2016). CCR2 is responsible for
monocyte chemotaxis to tissue (Bartoli et al., 2001) and recent in-
vestigations have shown a significant role of CCL2-CCR2 interaction in
M2 macrophage polarization (Sierra-Filardi et al., 2014;Deci et al.,
2018). To date, only one study has examined the monocyte CCR2
response to acute aerobic exercise (Okutsu et al., 2008). While in-
vestigators did not observe a significant change in monocyte CCR2
expression, it is important to note that the study did not examine po-
tential physical activity related differences among participants, nor did
the study differentiate CCR2 expression amongst monocyte subsets.
Importantly, aerobic training studies in both human and animal models
have shown the beneficial impact of exercise on macrophage polarization
in skeletal muscle (Walton et al., 2019) and adipose tissue (Kawanishi
et al., 2010). However, to our knowledge, there are currently no studies
that have investigated the potential role of CCR2 on circulating primary
monocytes in human macrophage polarization following a single bout of
aerobic exercise. In addition, the impact of habitual MVPA on monocyte
subset CCR2 expression and macrophage polarization following an acute
bout of exercise is unknown. Although habitual MVPA has unquestion-
able protective benefits against the development of CAD, the mechanisms
responsible remain elusive. Therefore, the purpose of this investigation
was to determine the impact of prior moderate to vigorous physical ac-
tivity on monocyte subset CCR2 surface expression and macrophage
polarization following an acute bout of moderate intensity cycle
ergometry.
2. Methods
2.1. Experimental design
Twenty four healthy women and men volunteered to participant in
the study. Physical activity levels were determined by scores from the
long form International Physical Activity Questionnaire (IPAQ)
(Hagstromer et al., 2006). Inclusion criteria consisted of normal body
mass index (18.5–24.9 kg/m2), 18–30 years of age, and normal per-
centage of body fat (males: 3–25%; females: 10–30%) (Jeukendrup and
Gleeson, 2010). Exclusion criteria consisted of tobacco use and use of
medications that may have impacted metabolism. In order to limit the
impact of sex hormones on monocytes, submaximal testing was per-
formed during the early follicular phase (first 7 days) of the menstrual
cycle for all female participants. All participants were instructed to limit
their physical activity 3 days prior to testing. Moderate to vigorous
physical activity (MVPA) was used to classify subjects into high physi-
cally active (HIACT: n ¼12; MVPA: 1500 MET min/wk) and low
physically active (LOACT: n ¼12; MVPA: <600 MET min/wk) groups.
Groups were chosen to differentiate between sufficient levels of physical
activity for optimal health benefits (HIACT) and some activity but not
sufficient for health benefits (LOACT), while simultaneously excluding
sedentary individuals (Sj€
ostr€
om et al., 2006). Macrophage phenotypes
exist on a broad spectrum, with M1 and M2 phenotypes representing
pro-inflammatory and anti-inflammatory extremes, respectively. Healthy
young men and women tend to have homogenous immune profiles,
regardless of fitness status (Beiter et al., 1985). Therefore, in order to
clearly determine the potential impact of acute exercise on macrophage
polarization we chose to focus on M1/M2 macrophage extremes. Study
procedures were approved by the Virginia Commonwealth Institutional
Review board (IRB#HM200008223) and all participants signed an
informed consent, volunteering to participate in the study.
2.2. Body composition testing
Participants arrived at the Virginia Commonwealth University Exer-
cise Physiology Research Laboratory (EPRL) between 7:00–8:00 a.m.
following an overnight fast, where height and weight were assessed.
Body composition was then assessed using air displacement plethys-
mography (BodPod, Cosmed, Rome, Italy) according to the manufac-
ture’s recommendations. Briefly, the BodPod was calibrated daily
according to manufacturer’s instructions and participants were instruc-
ted to wear minimal tight fitting clothing, remove all jewelry, and place
their hair under a swim cap. Participants were then seated within the
BodPod and two consistent (1%) body volume measurements were
taken. Measurements were entered into the manufacturer’s software and
percentage of fat mass was calculated using the Siri equation (Siri, 1961).
2.3. Peak graded exercise testing
Following analysis of body composition, participants were fitted with
a chest strap heart rate monitor (Polar Electro Inc., New York, USA) and
asked to sit quietly for 5 min. Resting heart rate was recorded and blood
pressure was manually assessed by an experienced technician using a
sphygmomanometer and stethoscope. Standard gas and volume for the
metabolic measurement system (TrueOne 2400, ParvoMedics, UT, USA)
were calibrated daily according to manufacturer’s instructions. The
acceptable percent change for calibrations was <1%. Participants were
seated on an electronically braked cycle ergometer (Ergoselect 100,
Ergoline, Bitz, Germany) and connected to the metabolic measurement
system in order to perform gas exchange analysis. Before beginning ex-
ercise, the test protocol was explained and a resting blood lactate mea-
surement was obtained from a finger stick blood sample using a blood
lactate analyzer (Lactate Scout þ, EKF Diagnostics, Cardiff, England).
Three minutes of pre-exercise data was collected in order to ensure gas
exchange measurements were within acceptable physiological ranges
(Mezzani, 2017). Following the rest period, participants entered a low
intensity warm-up stage where they were instructed to pedal at a cadence
of 50–100 RPM against a constant workload (Men: 50W; Women: 25W)
(Denadai et al., 2005). After completion of the warm-up stage, the
workload was consistently increased (Men: 25 W/min; Women: 15
A.M. Blanks et al. Brain, Behavior, &Immunity - Health xxx (xxxx) xxx
2
W/min) until volitional fatigue (Zhang et al., 1991;Albouaini et al.,
2007). Peak effort during the test was determined if a participant reached
three of the following criteria: peak heart rate 10 beats of age predicted
maximal heart rate (220-age), a rating of perceived exertion 17 on the
Borg scale, blood lactate 8 mmol/L, and a respiratory exchange ratio >
1.1 (Edvardsen et al., 2014).
2.4. Submaximal exercise testing
Participants were asked to return to the EPRL at least 3 days following
peak graded exercise testing. Again, participants were instructed to limit
their physical activity 3 days prior to testing and to fast overnight. Par-
ticipants’weight was assessed and they were fitted with a chest strap
heart rate monitor. In order minimize the impact of stress hormones and
cardiovascular parameters on immune function, prior to exercise par-
ticipants were seated and asked to rest quietly for 30 min (Hill et al.,
2008;Gu et al., 1999;Riou et al., 2007). Resting heart rate was then
recorded and blood pressure was manually assessed. A pre-exercise blood
sample (PRE) was obtained from an antecubital vein following standard
venipuncture guidelines (In:WHO guidelines, 2010). Venous blood was
obtained in two 10 mL blood collection tubes coated with sodium hep-
arin and one 10 mL serum separator tube (SST) (BD Vaccutainer, Becton,
Dickinson and Company, NJ, USA). The exercise testing procedure was
explained to the participants and they were then seated on the same cycle
ergometer and connected to the same metabolic measurement system
used for peak exercise testing. Three minutes of resting gas exchange data
was collected. Participants then performed a 3 min warm-up period
identical to the peak exercise test warm-up. The warm-up workload
(Men: 25 W/min; Women: 15 W/min) was subtracted from the measured
workload at 60% of VO
2peak
and the difference was divided by 5. This
calculation provided a value that was used to increase the workload in
equal increments each minute following warm-up until participants
reached a workload corresponding to 60% of VO
2peak
. Participants
maintained this workload for 25 min. If necessary, participants’work-
load was adjusted in order to maintain 60% of VO
2peak
. Blood lactate was
measured from a finger stick blood sample every 5 min to ensure par-
ticipants were below lactate threshold.
2.5. Sample processing and whole blood flow cytometry staining
Immediately following completion of the submaximal exercise test,
venous blood was obtained in two 10 mL sodium heparin tubes and one
10 mL SST tube (POST). Subsequent to POST venipuncture, participants
were asked remain fasted while sitting in the EPRL, and to avoid
engaging in activities that may have been mentally stressful (exam
studying, work deadline, etc.) in order to limit the impact of stress on
immune function. To assess the time course of the monocyte response,
additional venous blood samples identical to PRE and POST were ob-
tained 1 h (1H) and 2 h (2H) following POST measures. PRE and POST
blood samples were processed together. Briefly, 200
μ
L of whole blood
was removed from each tube and placed into 2 mL microcentrifuge tubes
(Safe-lock, Eppendorf, Hamburg, Germany). Whole blood was washed
once using 1.8 mL of freshly prepared flow cytometry staining buffer (1
PBS þ4% FBS). Blood samples were centrifuged at 1000 G for 10 min
and supernatant was aspirated and discarded. Careful attention was paid
not to disturb the buffy coat. Next, 1.8 mL of freshly prepared commercial
lyse/fix buffer (BD Phosflow Lyse/fix, Becton, Dickinson and Company)
was added and blood was incubated in a water bath at 37 C for 10 min in
order to lyse erythrocytes. Tubes were centrifuged at 600 G for 10 min,
supernatant was decanted and discarded, and cells were again washed
with 1 mL of staining buffer. Supernatant was decanted and cells were
suspended in 1 mL of freshly prepared commercial permeabilization
buffer (BD perm/wash buffer, Becton, Dickinson and Company) and
incubated at room temperature for 20 min. Cells were centrifuged for 10
min at 600 G and supernatant was decanted and discarded. Cells were
washed once using 1 mL of permeabilization buffer and suspended in
200
μ
L of permeabilization buffer. In order to block non-specific binding
of Fcγreceptors on myeloid cells, 5
μ
L of commercial Fc block (Human
TruStain FxX, Biolegend, CA, USA) was added to each tube and tubes
were incubated at room temperature for 10 min. To identity monocytes,
antibodies against CD14 (FITC conjugated anti-human antibody, clone:
M5E2, 0.5
μ
L/test, Biolegend), CD16 (APC conjugated anti-human anti-
body, clone: 3G8, 5
μ
L/test, Biolegend), CCR2 (PE conjugated anti-
human antibody, clone: K036C2, 2.5
μ
L/test, Biolegend) were added at
optimal concentrations as determined by previous titration experiments
and incubated at room temperature protected from light for 1 h. Cells
were washed twice using 3 mL of permeabilization buffer, suspended in
500
μ
L of permeabilization buffer, and stored at 4 C protected from light
until flow cytometry analysis. Flow cytometry staining was repeated in
an identical fashion for 1H and 2H blood samples. All analyses were
performed 3 days subsequent to processing for all whole blood samples.
2.6. Macrophage culture &flow cytometry staining
Following the removal of blood used for flow cytometry staining, 18
mL of heparinized blood was carefully layered onto 16 mL of room
temperature Hisotopaque 1077 (Sigma-Alrdich, MO, USA). Samples were
centrifuged at 600 G for 20 min. The top plasma layer was carefully
aspirated and stored at 80 C. The PBMC layer was collected using a
micropipette and washed twice using sterile PBS at 1000 G for 10 min.
In order to facilitate platelet removal, cells were washed using PBS þ1%
FBS and centrifuged at 200 G for 15 min. Cells were washed once more
with sterile PBS at 1000 G for 10 min. PBMCs were counted and 200
μ
L
of cell suspension was plated in duplicate wells at a concentration of 5
10
6
cells/mL in a 48 well tissue culture treated microplate (Corning
Incorporated, MA, USA). Cultures were placed into an incubator at 37 C
with 5% CO
2
for 2 h in order to allow monocytes to adhere. Cultures were
removed from the incubator, the cell culture supernatant was aspirated
and discarded, and plates were washed with sterile PBS at 1000 G for 5
min 200
μ
Lof37C complete culture media (DMEMþ1% pen-
strepþ20% autologous serum) was added to each well and plates were
returned to the incubator. Culture media was aspirated and replaced with
complete media every 2–3 days for a total of 7 days.
Following the 7 day culture, cell cultures were washed with PBS and
macrophages were released from the plastic by incubating wells with
300
μ
L of cell detachment solution (Accutase, Innovative Cell Technol-
ogies, Inc., CA, USA) at room temperature for 25 min (Davies et al.,
2017). Supernatant was collected and transferred to 2 mL micro-
centrifuge tubes. In order to assess cell viability, macrophages were
stained using a viability dye (Zombie Aqua Fixable Viability Kit, Bio-
legend) at a 1:500 concentration for 20 min. Cells were washed once
using staining buffer and stained using antibodies against CCR2, the M2
marker CD206 (PE/Cy5 conjugated anti-human CD206 (MMR) antibody,
clone: 15–2, 5
μ
L/test, Biolegend), and the M1 marker CD86 (Alexa Fluor
647 conjugated anti-human CD86 antibody, clone: IT2.2, 2.5
μ
L/test,
Biolegend) in identical fashion as whole blood samples. Macrophages
were analyzed immediately following antibody staining.
2.7. Flow cytometry analysis
All flow cytometry analyses were performed on a FACSCelesta (Bec-
ton, Dickinson and Company, NJ, USA) within the John Ryan Laboratory
at VCU. Flow cytometer setup and tracking was performed daily. Flur-
ochrome compensation was performed using unstained controls and
compensation beads (Ultracomp ebeads compensation beads, Thermo-
Fisher, MA, USA) stained with the antibodies being used in the experi-
ment. Doublet cells were gated out using a dot plot display of forward
scatter area versus forward scatter height (Fig. 1A). Following doublet
gating, monocytes were initially determined and gated based on forward
and side light scatter profiles and 2000 events were collected. An intra-
cellular staining buffer (Intracellular Staining Permeabilization Wash
Buffer, Biolegend, San Diego, CA) for markers not included in this
A.M. Blanks et al. Brain, Behavior, &Immunity - Health xxx (xxxx) xxx
3
investigation was used. When compared to standard flow cytometry
staining buffer (1PBS þ4% FBS), this buffer altered the cell light scatter
profile but did not impact receptor expression (pilot data not shown). In
order to ensure the inclusion of all monocyte events, the monocyte
scatter gate was widened (Fig. 1B). Monocytes were confirmed and gated
into subset quadrants using a dot plot of CD14 versus CD16 (Fig. 1C).
Macrophages were gated in a similar fashion. Briefly, doublet cells were
gated out, a gate was set for live cells (Fig. 1D) and macrophages were
gated based on scatter profile (Figs. 1E), and 2000 events were collected.
Adequate blocking of Fcγreceptors was assessed using appropriately
matched isotype controls (Biolegend) and receptor positivity was deter-
mined using fluorescence minus one controls. Histogram analysis was
then performed to analyze receptor expression within each monocyte and
macrophage subset (Fig. 1F). Monocyte and macrophage expression is
reported as mean fluorescent intensity in arbitrary units of fluorescence
(AUF).
2.8. Statistical analysis
Demographics of the study participants were compared using
descriptive statistics and independent samples t-tests. Due to the influ-
ence of blood pressure on monocyte adhesion and diapedesis, mean
arterial pressure (MAP) was analyzed as a covariate (Riou et al., 2007;
Tropea et al., 1996). Two-way analysis of covariance factorial (group x
time ANCOVAs) with Bonferroni adjustments were used to determine
differences in monocyte subset CCR2 expression, percentage of mono-
cytes positively expressing CCR2 (CCR2
þ
), and percentage of monocyte
subsets between and within groups (HIACT &LOACT) across all time
points. Two-way (group x time) ANCOVAs were used to determine dif-
ferences in macrophage CD206, CD86, CCR2 expression, and percentage
of macrophages positively expressing the aforementioned markers
(CD206
þ
, CD86
þ
, CCR2
þ
). Effect sizes (partial eta squared [
η
_P^2]) are
reported for the interaction terms of the ANOVA, where values of 0.01,
Fig. 1. Doublet cells were gated out using forward scatter height (FSC–H) and forward scatter area (FSC-A) (A). Monocytes were determined by side scatter area (SSC-
A) and FSC-A (B). Monocyte subsets were gated based on expression of CD14 and CD16 (C). Macrophage viability (D) was assessed and macrophages were gatedby
SSC-A and FSC-A (E). Monocyte CCR2 and macrophage CCR2, CD86, and CD206 were assessed using histogram analysis (F). Fluorescence minus one control samples
(purple) were used to set gates for positive receptor expression (red). (For interpretation of the references to colour in this figure legend, the reader is referred to the
Web version of this article.)
A.M. Blanks et al. Brain, Behavior, &Immunity - Health xxx (xxxx) xxx
4
0.06, and 0.14 correspond to small, medium, and large effects, respec-
tively (Cohen, 1988). Statistical analyses were performed with SPSS
Version 24 software (IBM) and data are presented as mean standard
error of the mean (SEM). The level of significance for all tests was set a
priori at
α
0.05.
3. Results
Participant demographics are presented in Table 1. By design, a sta-
tistically significant difference was observed in physical activity. As
physical activity has been strongly associated with VO
2peak
(Schembre
and Riebe, 2011), a significantly different VO
2peak
was observed between
the HIACT and LOACT groups. No other significant differences were
observed between groups.
3.1. Monocyte CCR2 expression &subset response
Pre-exercise CCR2 expression was not different between groups in any
monocyte subset. Classical (Fig. 2A) and non-classical (Fig. 2C) CCR2
expression was not changed at any time point in either group following
exercise (p >0.05). A group by time effect was observed for intermediate
monocyte CCR2 expression (p ¼0.040,
η
¼0.123) in response to exercise
(Fig. 2B). Individual values for PRE and POST intermediate monocyte
CCR2 expression are presented in Fig. 3A&B. In the HIACT group in-
termediate CCR2 expression was reduced immediately post-exercise (PRE:
11409.0 1084.0 vs. POST: 9524.3 1062.4 AUF; p ¼0.034) (Fig. 3A).
Intermediate CCR2 expression returned to baseline at 1H (1H: 11847.0
1191.6 AUF) (Fig. 2B). Intermediate CCR2 expression was not impacted
by exercise in the LOACT group (p >0.05) (Fig. 2B). The percentage of
CCR2
þ
monocytes was not changed in any monocyte subset in either
group following exercise (p >0.05). For all subjects as whole, a time effect
(p ¼0.020,
η
¼0.14) was observed for the percentage of classical
monocytes (1H: 83.5 2.3 vs. 2H: 75.8 3.1%, p ¼0.008) (Fig. 4).
3.2. Macrophage polarization
No pre-exercise differences were observed between groups in the
percentage of CD86
þ
, CD206
þ
,orCCR2
þ
macrophages (p >0.05). Addi-
tionally, no difference was observed in receptor expression at PRE. A group
by time effect was not observed in macrophage polarization (p >0.05);
however, a significant difference was found between groups (p ¼0.049,
η
¼0.199) in the percentage of CD206
þ
macrophages at 1H (HIACT: 67.2
5.6 vs. LOACT: 50.1 5.2%; p ¼0.040) (Fig. 5). No differences were
observed in the M1/M2 ratio at any time point (p >0.05).
3.3. Relationships between monocytes ¯ophages
When all participants were analyzed together, macrophage CD206
expression at 1H was positively associated with non-classical monocyte
CCR2 expression at PRE (r ¼0.446, p ¼0.043) as well as POST (r ¼
0.464, p ¼0.034). The percentage of CCR2
þ
non-classical monocytes at
PRE was negatively associated with macrophage CD86 expression at PRE
(r ¼-0.415, p ¼0.028). Immediately post-exercise, macrophage CD86
expression was negatively associated with the percentages of classical (r
¼-0.436, p ¼0.33) and non-classical (r ¼-0.455, p ¼0.025) CCR2
þ
monocytes at POST. When expressed as a percentage of total monocytes,
the percentage of the classical subset at POST was negatively associated
with M1/M2 macrophage ratio at POST (r ¼-0.405, p ¼0.049), the
percentage of the intermediate subset at POST was negatively associated
with M1/M2 macrophage ratio at 2H (r ¼0.437, p ¼0.042), and the
percentage of the non-classical subset at PRE was associated with M1/M2
macrophage ratio at PRE (r ¼0.408, p ¼0.048).
4. Discussion
The purpose of the present study was to determine if monocyte subset
CCR2 surface expression and macrophage polarization in response to an
acute bout of moderate intensity exercise are different between high
physically active compared to low physically active individuals. Findings
Table 1
Participant demographics for high physically active (HIACT) and low physically
inactive (LOACT) groups. MVPA (Moderate-to-vigorous physical activity), MAP
(Mean arterial pressure). Data are presented as the mean standard error of the
mean. *p <0.05 between groups; Independent samples t-test.
Variable HIACT (n ¼12) LOACT (n ¼12) p-value
Sex (F/M) 6/6 6/6 n/a
Age (yrs) 23.8 0.7 22.8 0.9 0.396
Height (cm) 170.6 2.3 170.1 3.6 0.900
Weight (kg) 65.5 2.5 64.2 3.6 0.769
Body Mass Index (kg/m
2
) 22.5 0.4 22.4 0.6 0.932
Body Fat (%) 16.3 1.9 18.6 1.9 0.395
MVPA (METmin/wk) 3848.3 593.3 378.1 65.5 <0.001*
VO
2peak
(L min
1
) 3.0 0.3 2.1 0.2 0.007*
VO
2peak
(mL kg min
1
) 45.0 2.5 32.5 1.6 <0.001*
MAP (mmHg) 84.9 2.4 89.9 1.9 0.117
Blanks, AM Physical Activity, Monocyte CCR2, and M1/M2 Macrophages.
Fig. 2. Time course of the mean fluorescent intensity (MFI) of CCR2 on the
classical (A), intermediate (B), and non-classical (C) monocyte subsets in high
physically active (HIACT) and physically low active (LOACT) individuals. 2 4
repeated measures ANCOVA. *p <0.05 PRE vs. POST within HIACT group; 2
4 repeated measures ANCOVA.
A.M. Blanks et al. Brain, Behavior, &Immunity - Health xxx (xxxx) xxx
5
of the present study demonstrate that an acute bout of exercise elicits a
monocyte response in both high and low physically active individuals.
Although acute exercise elicits responses in both high and low active
individuals, intermediate monocyte CCR2 is reduced and macrophage
CD206 is unchanged in highly active individuals, as compared to low
active individuals. To our knowledge, this is first study to demonstrate
that monocyte CCR2 expression and macrophage polarization responses
to a single session of moderate intensity exercise are beneficially
impacted by high levels of prior physical activity.
The surface expression of CCR2 on the intermediate monocyte subset
was reduced immediately post-exercise in the HIACT group. Previous
investigations have consistently shown that exercise at or above 60% of
VO
2peak
in young healthy individuals causes an increase in plasma
cortisol concentrations above resting levels (Budde et al., 2015).
Although cortisol levels were not measured in the current investigation, a
previous investigation of the monocyte CCR2 response to exercise
showed that incubation of monocytes with post-exercise serum led to a
cortisol dependent increase in CCR2 surface expression (Okutsu et al.,
2008). Therefore, in the LOACT group it is plausible that
exercise-induced cortisol did in fact increase CCR2 expression, however
this elevation may have been balanced by the ligand-receptor internali-
zation that occurs when CCL2 binds to CCR2 (Volpe et al., 2012). Acute
increases in cortisol are necessary for a proper immune response
(Dhabhar, 2002), however, chronic elevations of cortisol elicit immu-
nosuppression via leukocyte desensitization to cortisol (Coutinho and
Chapman, 2011). Although training status does not impact cortisol
release in response to acute exercise in young healthy adults (Duclos
et al., 1997), repeated exercise bouts may specifically reduce the
response of pro-inflammatory monocytes to cortisol (Ehrchen et al.,
2007), without immunosuppression due to the production of IL-6 and
IL-10 that occur with exercise (Tsianakas et al., 2012;Pedersen et al.,
2001;Cabral-Santos et al., 2019). Therefore, in the HIACT group, it does
not appear that cortisol increased CCR2 expression and the observed
post-exercise reductions in intermediate monocyte CCR2 expression
were likely due to CCR2-CCL2 binding and internalization. Activated
intermediate monocytes are pro-inflammatory in nature and contribute
to the pro-inflammatory microenvironment which elicits monocytes to
differentiate into pro-inflammatory M1 macrophages (Wong et al., 2011;
Italiani and Boraschi, 2014). This relationship was evidenced by the
positive association between the percentage of intermediate monocytes
immediately post-exercise and M1/M2 macrophage ratio 2 h following
exercise. Monocyte CCR2 binding to CCL2 stimulates chemotaxis along a
chemical ligand gradient and receptor-ligand internalization acts to clear
CCL2 from circulation, thereby reducing activation of additional cells in
circulation (Volpe et al., 2012). As CCR2 expression has been shown to
directly impact monocyte chemotaxis (Fantuzzi et al., 1999), the lower
post-exercise intermediate monocyte CCR2 expression observed in the
HIACT group blunts the acute pro-inflammatory response to exercise and
likely contributes to reduced M1 macrophage polarization in tissue.
Taken together, these data demonstrate a potential mechanism through
which regular physical activity acts to prevent CAD.
The percentage of macrophages expressing the anti-inflammatory M2
marker, CD206, was greater in the HIACT group compared to the LOACT
group 1 h following exercise. Although a group by time effect was not
observed, the percentage of CD206 positive macrophages appeared to be
lower following exercise in the LOACT group while remaining un-
changed in the HIACT group. Sustained inflammation, creates a pro-
inflammatory microenvironment which leads to pro-inflammatory acti-
vation of monocytes, preferential differentiation to pro-inflammatory M1
macrophages, and macrophage phenotype switching from M2 to M1
(Moore and Tabas, 2011;Moore et al., 2013). Together, these
pro-inflammatory alterations skew macrophage balance towards the M1
phenotype, thereby leading to the pathogenesis and progression of CAD
(Moore et al., 2013). Acute bouts of exercise have been shown to elicit an
acute pro-inflammatory response which is necessary for muscle repair
following exercise (Suzuki, 2018;Yang and Hu, 2018). This
Fig. 3. Individual values of intermediate monocyte CCR2 MFI in HIACT (B) and
LOACT (C) groups. *p <0.05 PRE vs. POST within HIACT group; 2 4 repeated
measures ANCOVA.
Fig. 4. Time course of the percentages of classical monocytes in high physically
active (HIACT), low physically active (LOACT), and both groups analyzed
together (ALL). *p <0.05 1H vs. 2H time effect for ALL; 2 4 repeated mea-
sures ANCOVA.
Fig. 5. Time course of the percentage of macrophages expressing CD206 in high
physically active (HIACT) and low physically active (LOACT) individuals. *p <
0.05 between groups; 2 4 repeated measures ANCOVA.
A.M. Blanks et al. Brain, Behavior, &Immunity - Health xxx (xxxx) xxx
6
pro-inflammatory response is followed by an anti-inflammatory response
which acts to quench inflammation (Suzuki, 2018). Although an exercise
induced cytokine response has been observed in both trained and un-
trained individuals, the magnitude and time course is different (Schild
et al., 2016). The cytokine microenvironment in which macrophages are
exposed to will impact macrophage polarization (Wang et al., 2014) and
although the cytokine response to exercise was not assessed in the cur-
rent investigation, it is likely that a greater magnitude and more rapid
time course of anti-inflammatory cytokine production led to a favorable
anti-inflammatory microenvironment and preservation of M2 macro-
phage polarization in the HIACT group. whereas pro-inflammatory cy-
tokines reduced M2 macrophage polarization in the LOACT group. In
healthy individuals, exercise eventually leads to an anti-inflammatory
response (Brown et al., 2015), which likely occurred at the 2 h time
point in the LOACT group, thereby returning M2 macrophage polariza-
tion to baseline. Taken together, these data suggest that physical activity
status positively impacts CAD risk by preserving anti-inflammatory M2
macrophage polarization following an acute bout of exercise.
In addition to its role in monocyte chemotaxis, CCR2 activation has
been shown to play a role in macrophage polarization (Sierra-Filardi
et al., 2014;Deci et al., 2018). Although CCR2 expression was not altered
in response to exercise in classical and non-classical monocyte subsets,
the percentage of CCR2
þ
classical monocytes was negatively associated
with macrophage expression of the M1 marker, CD86. Under homeo-
static conditions, classical monocytes are considered to be
anti-inflammatory and contribute to an anti-inflammatory microenvi-
ronment, which elicits monocyte differentiation to the M2 macrophage
phenotype (Mukherjee et al., 2015;Boyette et al., 2017;Jakubzick et al.,
2013). The CCL2 response to muscle damage induced by acute exercise is
equivocal (Lu et al., 2011;Peake et al., 1985). Although CCL2 was not
assessed in the current investigation, none of the participants were
trained cyclist, therefore participants in both groups likely experienced
exercise-induced muscle damage and a subsequent increase in plasma
CCL2 concentrations (Burt et al., 2012;Fredsted et al., 2008). Therefore,
greater percentages of classical monocytes expressing CCR2 led to more
monocytes being activated by CCL2, reduced M1 macrophage polariza-
tion, and a more favorable M1/M2 macrophage ratio as a result. More-
over, non-classical monocyte CCR2 expression at PRE and POST was
positively associated with macrophage expression of the M2 marker,
CD206. The percentage of pre-exercise CCR2
þ
non-classical monocytes
was negatively associated with macrophage CD86 expression prior to
exercise, further supporting the involvement of CCR2 in macrophage
polarization. The pro-inflammatory nature of the non-classical monocyte
subset is thought to be due to senescence (Ong et al., 2018). Aging is
associated with increased CVD risk due to a heightened inflammatory
status, known as “inflammaging”, which includes increased production
of CCL2 (Franceschi et al., 2018;Antonelli et al., 2006). Increased CCL2
may be a compensatory mechanism to cope with the expansion of the
non-classical monocyte subset, which express low levels of CCR2 (Ong
et al., 2018). Recent investigations support the hypothesis that regular
physical activity reduces inflammaging (Flynn et al., 2019). Although the
mechanisms responsible for physical activity blunting of inflammaging
are unclear, it is plausible to suggest that chronic physical activity slows
monocyte aging by preserving CCR2 expression, thereby altering the
phenotype of non-classical monocytes towards a less inflammatory
phenotype similar to that of younger classical and intermediate mono-
cytes. Taken together, these data demonstrate a significant role of CCR2
expression in macrophage polarization for all monocyte subsets. More-
over, these data suggest that physical activity may beneficially impact the
relationship between monocyte CCR2 expression and macrophage
polarization.
5. Conclusion
This is the first study to demonstrate the impact of physical activity on
monocyte CCR2 expression and in-vitro macrophage polarization
following an acute bout of moderate intensity exercise. Perhaps most
importantly, the monocyte and macrophage responses to acute exercise
appear to be different between high physically active and low active
individuals. Although the current investigation examined monocyte/
macrophage responses to a single bout of exercise, based on the study
findings, it is likely that repeated bouts of moderate to vigorous physical
activity would lead to long term adaptations in low physically active
individuals and improve their monocyte/macrophage response, similar
to that of high physically active individuals. Future studies are warranted
to investigate the potential impact of repeated bouts of physical activity
on the monocyte and macrophage response. Nonetheless, the findings
from the current study suggest that physical activity positively impacts
both monocytes and macrophages following acute moderate intensity
exercise and more importantly, this impact may contribute to the pre-
vention of coronary artery disease.
Funding
This research did not receive any specific grant from funding agencies
in the public, commercial, or not-for-profit sectors.
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