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Copyright © 2018 American College of Sports Medicine
Resistance Exercise Selectively Mobilizes Monocyte Subsets:
Role of Polyphenols
Adam R. Jajtner1, Jeremy R. Townsend2, Kyle S. Beyer3, Alyssa N. Varanoske4,
David D. Church4, Leonardo P. Oliveira4, Kelli A. Herrlinger5, Shlomit Radom-Aizik6,
David H. Fukuda4, Jeffrey R. Stout4, Jay R. Hoffman4
1Department of Exercise Physiology, Kent State University, Kent, OH; 2Exercise and Nutrition
Science, Lipscomb University, Nashville, TN; 3Department of Exercise Science, Bloomsburg
University, Bloomsburg, PA; 4Institute of Exercise Physiology and Wellness, University of
Central Florida, Orlando, FL; 5Kemin Foods, L.C., Des Moines, IA; 6Pediatric Exercise and
Genomics Research Center (PERC), University of California – Irvine, Irvine, CA
Accepted for Publication: 18 June 2018
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RESISTANCE EXERCISE SELECTIVELY MOBILIZES MONOCYTE
SUBSETS: ROLE OF POLYPHENOLS
Adam R. Jajtner1, Jeremy R. Townsend2, Kyle S. Beyer3, Alyssa N. Varanoske4,
David D. Church4, Leonardo P. Oliveira4, Kelli A. Herrlinger5, Shlomit Radom-Aizik6,
David H. Fukuda4, Jeffrey R. Stout4, Jay R. Hoffman4
1Department of Exercise Physiology, Kent State University, Kent, OH; 2Exercise and Nutrition
Science, Lipscomb University, Nashville, TN; 3Department of Exercise Science, Bloomsburg
University, Bloomsburg, PA; 4Institute of Exercise Physiology and Wellness, University of
Central Florida, Orlando, FL; 5Kemin Foods, L.C., Des Moines, IA; 6Pediatric Exercise and
Genomics Research Center (PERC), University of California – Irvine, Irvine, CA
Author Contributions: Conception and design of research: ARJ, JRH, JRT, KSB, DDC, KAH,
JRS; Acquisition of data: ARJ, JRT, KSB, ANV, DDC, LPO; Data analysis and interpretation:
ARJ, JRH, DDC, DHF; Manuscript draft and revision: ARJ, JRH, KAH SRA, DHF, JRS;
Approval of final version: ARJ, JRH, JRT, KSB, ANV, DDC, LPO, KAH, SRA, DHF, JRS.
Clinical Trial Registration: NCT02442245
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Medicine & Science in Sports & Exercise, Publish Ahead of Print
DOI: 10.1249/MSS.0000000000001703
Copyright © 2018 by the American College of Sports Medicine. Unauthorized reproduction of this article is prohibited.
Corresponding Author:
Adam R. Jajtner, PhD.
Department of Exercise Physiology; Kent State University
350 Midway Dr.; Kent, OH 44242
Phone: 330-672-0212; Fax: 330-672-2250
ajajtner@kent.edu
Funding: Kemin Foods, L.C.
Conflict of Interest: KAH is employed by Kemin Foods, LC. All other authors report no actual
or potential conflicts of interest.
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ABSTRACT
Purpose: To examine the impact of polyphenol supplementation on the recruitment,
mobilization and activation of monocyte subsets following resistance exercise. Methods:
Thirty-eight recreationally active males (22.1 ± 3.1 yrs; 173.9 ± 7.9 cm; 77.8 ± 14.5 kg) were
assigned to 28-days of polyphenol supplementation (PPB), placebo (PL) or control (CON).
Blood samples were obtained before (PRE), immediately-(IP), one-(1H), five-(5H), 24-(24H)
and 48-(48H) hours post-resistance exercise (PPB/PL) or rest (CON). Fine-needle biopsies were
obtained from the vastus lateralis at PRE, 1H, 5H and 48H. Circulating concentrations of
macrophage chemoattractant protein-1 (MCP-1) and fractalkine, as well as intramuscular MCP-1
were analyzed via multiplex assay. Changes in the proportions and expression of CD11b on
monocyte subsets were assessed via flow cytometry. Results: Circulating MCP-1 increased in
PPB and PL at IP with further increases at 5H. Intramuscular MCP-1 was increased at 1H, 5H
and 48H in all groups. Classical monocyte proportions were reduced in PPB and PL at IP, and
increased at 1H. Nonclassical monocytes were increased in PPB and PL at IP, while
intermediate monocytes were increased at IP, and reduced at 1H. Intermediate monocytes were
increased in PPB at 24H and 48H. CD11b expression was reduced on PPB compared to PL and
CON at PRE on intermediate and nonclassical monocytes. Conclusions: Resistance exercise
may elicit selective mobilization of intermediate monocytes at 24H and 48H, which may be
mediated by tissue damage. Additionally, polyphenol supplementation may suppress CD11b
expression on monocyte subsets at rest.
Key Words: Intermediate Monocyte, Inflammation, CD11b/CD18, Monocyte Chemoattractant
Protein-1 (MCP-1)
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INTRODUCTION
When performed at a sufficient intensity, resistance exercise is known to induce skeletal
muscle damage (1) and an elevation in markers of oxidative stress (2). As a result, antioxidant
supplementation has been used to potentially reduce this exercise-induced stress (3).
Polyphenols are micronutrients with antioxidant properties that are common in many foods
including tea (4), and have also been examined for their potential benefits associated with
recovery from exercise induced muscle damage (3,5,6). Previous studies have shown favorable
results with regard to improved maintenance of muscular force, and reduced markers of muscle
damage following resistance exercise with polyphenol supplementation (3). However, recovery
from exercise-induced muscle damage, as well as the adaptation to exercise training, is more
complex than simply limiting the initial insult to skeletal muscle tissue.
An adequate resistance exercise initiates an inflammatory cascade that results in the
secretion of various chemotactic factors (7), which in turn recruit specific immune cells (8,9).
The two primary immune cells of interest are the neutrophil and monocyte/ macrophage, which
are recruited during three primary phases of recovery: preliminary, early and late (10). While
neutrophils are the predominant cell involved with the preliminary phase (10), the
monocyte/macrophage, is the primary cell of interest for both the early and late phases of
recovery (10).
Macrophages, which represent a developmental transition of circulating monocytes
within the tissue, can be subdivided into two primary subsets, M1 and M2 (11). M1
macrophages are most prominent during the early phase of recovery, while M2 macrophages are
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prominent during the late phase (10). Given that both subsets produce insulin-like growth factor
1 (IGF-1) (12), macrophages play a prominent role in skeletal muscle recovery (10). Moreover,
considering that M1 macrophages promote a cytokine milieu conducive to the proliferation of
myogenic precursor cells (MPC), and M2 macrophages promote MPC differentiation (13), the
timely transition between these phases may be of utmost importance during muscle adaptation
resulting from exercise stress.
Although disputed (13), the development of macrophage subsets may be directly linked
to the circulating monocyte subset of origin (14). Recently, monocytes have been formally
organized into a three subset paradigm (15), replacing the two subset model of classical and
nonclassical monocytes (16). Phenotypically, monocytes are identified based on their expression
of the lipopolysaccharide (LPS) receptor, cluster of differentiation (CD) 14, and the FcγRIIIa
receptor, CD16. Though both classical (CD14++/CD16-) and nonclassical (CD14+/CD16++)
monocytes have been suggested to give rise to M1 and M2 macrophages, respectively (13,14), a
formal role of intermediate (CD14++/CD16+) monocytes within the resolution of skeletal
muscle damage have yet to be identified.
Typically, the total monocyte population, which accounts for approximately 5-10% of the
total leukocyte population (17), consists of 80-90% classical, 5-10% intermediate and 5-10%
nonclassical monocytes (15,16). The proportions of these subsets are altered immediately
following exercise; characterized by reduced proportions of classical monocytes, with a
concomitant increase in the proportion of nonclassical monocytes (17,18). At one hour
following exercise, this modulation of monocyte subset proportions has been shown to return to
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baseline (17), or super-compensate (increased classical monocyte proportion, and reduced
nonclassical monocyte proportion) (18). To date, only one investigation has examined the
change in classical monocytes following resistance exercise, indicating increased proportions of
classical monocytes from 1- to 5-hours into recovery (19). However, this prior investigation (19)
did not examine the intermediate or nonclassical response to resistance exercise. We expect the
response of intermediate and nonclassical monocytes to be different following resistance
exercise compared to aerobic exercise due to increased muscle damage (6,20). To our
knowledge, no other investigations have characterized the intermediate or nonclassical monocyte
response to resistance exercise.
Recruitment of classical and nonclassical monocytes is accomplished primarily by
monocyte chemoattractant protein-1 (MCP-1) (11,16,21), and fractalkine (CX3CL1) (22),
respectively. Although existing data examining the post-resistance exercise response of these
specific cytokines is limited, elevations of both MCP-1 and CX3CL1 within skeletal muscle have
been reported following resistance exercise for up to four and two hours, respectively (8).
Furthermore, data involving MCP-1 in circulation has been conflicting, with both a post-exercise
increase (23), and decrease (24) demonstrated previously, while no investigation has examined
circulating CX3CL1 following resistance exercise.
In addition to changes in monocyte subset proportions and recruitment following
resistance exercise, changes in cellular activation of these cells have also been documented
(23,25). Complement receptor 3 (CR3) is a β2 integrin that is composed of CD11b and CD18,
and involved in the late phases of transendothelial migration to damaged tissue (26). Therefore,
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CD11b plays a crucial role in the transmigration of monocytes into damaged tissue. Previously,
polyphenol supplementation has resulted in decreases in CD11b expression at rest in individuals
at high risk for cardiovascular or metabolic disease (27). Furthermore, in vitro incubation of
monocytes and neutrophils in polyphenols has resulted in reduced expression of adhesion
molecules and reduced chemotaxis (28). Consequently, it is possible that polyphenols may limit
the expression of CD11b on monocytes during and following exercise.
To date, few investigations have examined CD11b or CR3 expression on monocytes
following resistance exercise or muscle damage, yielding little consensus of a time course of
elevated expression. Briefly, dynamic resistance exercise has been demonstrated to result in
increased expression of CD11b during the first hour of recovery (23,25), though following
exercise designed to elicit muscle damage, CD11b expression has been reported to both increase
(29) or remain unchanged (30). To our knowledge, no investigation has examined how
polyphenol supplementation may modify the post-exercise response.
Therefore, the primary purpose of this investigation was to examine the impact of
polyphenol supplementation on the overall recruitment of monocytes following resistance
exercise, in addition to the overall expression of CD11b on monocytes. Furthermore, we aimed
to detail the post-resistance exercise recruitment, modulation and activation of monocyte subsets.
We hypothesized that resistance exercise would result in a significant increase in the proportion
of nonclassical monocytes immediately following resistance exercise, and that classical
monocyte proportions would display a super-compensation at 1 hour that remains elevated for up
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to five hours into recovery. Furthermore, we anticipated polyphenol supplementation would
reduce the CD11b expression on monocytes following resistance exercise.
METHODS
Participants
Recreationally active, but non-resistance trained men (n = 38; 22.1 ± 3.1 y; 173.9 ± 7.9
cm; 77.8 ± 14.5 kg) volunteered to participate in this study. Sample size was determined based
on creatine kinase (CK) concentrations from a previous investigation utilizing the same
supplement (5). Following explanation of all procedures, benefits, and risks, participants gave
their informed written consent prior to participation, and all procedures were approved by the
New England Institutional Review Board. For inclusion in this investigation, participants had to
engage in less than 3 hours per week of planned exercise, have a body mass index of between
18.5 and 34.9 kg/m2, be free from physical limitations, and willing to maintain a normal diet
while abstaining from tea, alcohols and additional dietary supplements. No other restrictions
were placed on participants’ diets, though daily consumption was monitored and confirmed
similar between groups via daily food logs (6).
Study Design
For this randomized, placebo-controlled, between-subjects investigation, participants
were assigned to one of three groups: proprietary polyphenol blend (PPB), placebo (PL) and
control (CON). Participants reported to the Human Performance Laboratory for four days of
testing (Figure 1). Prior to the first day of testing, PPB and PL completed 28-days of
supplementation. During Day 1, participants completed one-repetition maximum (1-RM) testing
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of the squat, leg press and leg extension exercises. At least 72 hours later, participants returned
to the lab following a 12-hour fast and provided a resting blood sample and skeletal muscle
biopsy (PRE). Participants were then provided a small breakfast bar (Cal: 190, CHO: 19g,
Protein: 7g, Fat: 13g) with limited polyphenols (4), followed by the acute exercise protocol (PPB
and PL) or rest for one hour (CON). Participants provided blood samples immediately (IP), 1-
hour (1H) and 5-hours (5H) post-exercise, and skeletal muscle biopsies at 1H and 5H. After the
1H samples were obtained, participants were provided a small, standardized meal with limited
polyphenols (4) (Cal: 250, CHO: 34g, Protein: 14g, Fat: 6g). Participants returned to the lab in a
fasted state, and provided resting blood samples at 24-hours (24H) and 48-hours (48H) post-
exercise, in addition to a skeletal muscle biopsy at 48H.
[Insert Figure 1 Approximately Here]
Supplementing Protocol
Both groups were supplemented daily for 28 days with either a proprietary polyphenol
blend (PPB) or placebo (PL) (Kemin Foods, L.C., Des Moines, IA, USA). Both participants and
investigators were blinded to the actual group assignments. The PPB group consumed a blend of
water-extracted green and black tea (Camellia Sinensis) containing at minimum 40% total
polyphenols, 1.3% theaflavins, 5-8% epigallocatechin-3gallate (EGCG), 7-13% caffeine, 600
ppm manganese. The PL group consumed microcrystalline cellulose in capsules of similar shape
and size. All products were tested for toxins including heavy metals and pesticides by an
independent third party.
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During the supplementation period, participants reported to the lab between three and
five days per week to receive their supplement. Participants took one dose (1000 mg PPB, or
PL) under the supervision of a member of the research team, and then were provided their
remaining doses in individual containers (1000 mg PPB or PL for each additional time point).
Participants consumed two doses daily, for a total of 2000 mg of either PPB or PL, and were
asked to return all empty containers to the lab on their next visits to monitor compliance. A dose
was considered to be administered if the participant consumed the dose in front of the study staff,
or returned their empty container and verbally confirmed they had taken the dose.
Supplementation continued throughout the acute exercise protocol, and during the four days of
recovery. Participants that did not maintain 80% compliance for each phase of the study (the 28
days of supplementation, during the acute exercise protocol, and the recovery period from the
acute exercise) were removed from the analysis.
Acute Exercise Protocol
The acute exercise session was completed by PPB and PL, while CON rested for one
hour. The protocol designed to elicit muscle damage was preceded by a light warm-up identical
to the 1-RM session. Participants then completed six sets of 10 repetitions of the squat, as well
as four sets of 10 repetitions of the leg press and leg extension exercises. All exercises were
completed at 70% of the participant’s previously determined 1-RM, with 90 seconds of rest
between sets. If participants were unable to complete 10 repetitions, they were provided with
assistance, and the weight on subsequent sets was reduced. All testing sessions, including 1-
RM’s, were overseen by a Certified Strength and Conditioning Specialist to monitor adherence
to exercise technique.
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Blood Sampling
Blood samples were obtained at seven time points throughout the study (PRE, IP, 1H,
5H, 24H, and 48H). The PRE, IP and 1H blood samples were obtained using a Teflon cannula
placed in a superficial forearm vein using a three-way stopcock with a male luer lock adapter and
a plastic syringe. The cannula was maintained patent using an isotonic saline solution (Becton
Dickinson, Franklin Lakes, NJ). PRE and 1H blood samples were obtained following a 15-
minute equilibration period, while IP blood samples were taken within 5-min of exercise
cessation. The remaining time points (5H, 24H, and 48H) were obtained by a single-use
disposable needle with the subject in a supine position for at least 15 minutes prior to sampling.
Whole blood (20 ml) was collected in two Vacutainer® tubes (Becton Dickinson, Franklin
Lakes, NJ), one containing K2EDTA, and one containing no anti-clotting agents. Aliquots were
removed from the first tube for hematocrit and hemoglobin measures, as well as flow cytometry
analysis, while the second tube was allowed to clot for 30 minutes prior to being centrifuged at
3,000 x g for 15 minutes. The resulting plasma and serum were aliquoted and stored at -80°C for
later analysis.
Analysis of Circulating Analytes
Plasma concentrations of monocyte-chemoattractant protein-1 (MCP-1), and fractalkine
(CX3CL1) were analyzed via multiplex assay (EMD Millipore, Billerica, MA, USA). All
samples were thawed once and analyzed in duplicate by the same technician using the MagPix
(EMD Millipore), with an average coefficient of variation of 6.84%, and 7.18% for MCP-1, and
CX3CL1, respectively. Serum concentrations of myoglobin and CK were assessed as previously
reported (6).
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Fine Needle Skeletal Muscle Biopsy Procedure
Fine needle muscle biopsies were performed on the vastus lateralis muscle of the
participant’s dominant leg using a spring-loaded, reusable instrument with 14-gauge disposable
needles and a coaxial introducer (Argon Medical Devices Inc., Plano, TX, USA). Following
local anesthesia with 2 mL of 1% lidocaine applied into the subcutaneous tissue, a small incision
to the skin was made and an insertion cannula was placed perpendicular to the muscle until the
fascia was pierced (31). Each muscle sample was removed from the biopsy needle using a sterile
scalpel and was subsequently placed in a cryotube, rapidly frozen in liquid nitrogen, and stored
at -80˚C. A new incision was made for each time point, with approximately 2cm between all
sampling sites. All muscle biopsies were performed by a licensed medical physician.
Intramuscular Cytokine Protein Content
Sufficient sample was not obtained from nine participants (PPB = 2, PL = 3, CON = 4),
and therefore were not included in the intramuscular analysis. Tissue samples were thawed and
kept on ice for preparation and homogenization. A proprietary lysis buffer with protease
inhibitor (EMD Millipore, Billerica, MA, USA) was added to each sample at a rate of 500 uL per
10 mg of tissue. Samples were homogenized using a Teflon pestle and sonication (Branson,
Danbury, CT, USA). Tissue samples were then agitated for 10 minutes at 4°C and centrifuged at
10,000 x g for 5 minutes. The supernatant was then aspirated and used for analysis.
Total protein content was assessed using a detergent compatible (DC) protein assay kit
(Bio-Rad, Hercules, CA, USA), and samples were diluted to 0.8 – 1.2 mg/ml. The protein
content of MCP-1 was then assessed via multiplex assay (EMD Millipore, Billerica, MA, USA)
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per manufacturer’s guidelines, and normalized to the total protein content. To limit inter-assay
variance, all tissue samples were analyzed in duplicate in the same assay run by a single
technician, with an average coefficient of variation of 9.37%. Intramuscular cytokine protein
content is expressed in pg/μg total protein (8).
Monocyte Subset Preparation
Fresh, anti-coagulated (K2EDTA), whole blood (100 µl) was mixed with fluorescent-
conjugated monoclonal antibodies specific to CD11b-fluorescein isothiocyanate (FITC;
Biolegend, San Diego, CA, USA), CD66b-phycoerythrin (PE), CD14-PerCP Cy5.5 and CD16-
allophycocyanin (APC; BD Biosciences, San Jose, CA, USA) within 10 minutes of blood
sampling. Samples were mixed and incubated for 15 minutes in the dark, after which, the
samples were lysed with 2 ml of 1 x FACS lysing solution (BD Biosciences), mixed and
incubated in the dark for an additional 8 minutes. Following incubation, samples were
centrifuged at 300 x g for 8 minutes and washed with 2 ml of 1 x wash buffer containing 1%
fetal bovine serum (FBS) in a 1 x phosphate buffered saline (PBS) solution. Samples were
centrifuged again at 300 x g for 8 minutes, and the supernatant was removed. Samples were then
fixed in 300 µl of 2% paraformaldehyde in PBS.
Flow Cytometry Analysis
Cell preparations were acquired using an Accuri C6 flow cytometer (BD Accuri
Cytometers, Ann Arbor, MI, USA) equipped with two lasers providing excitation at 488 and 640
nm, and 4 band pass filters (FL1: 533/30; FL2: 585/40; FL3 670LP; FL4: 675/25). Events were
recorded based on size (FSC-A), complexity (SSC-A) and mean fluorescence intensity (MFI). A
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total of 200 µl were collected for each sample, with at least 8,000 CD14+ events. If 8,000
CD14+ events were not obtained, the sample was removed from analysis.
The analysis was completed using BD Accuri analysis software (BD Accuri Cytometers,
Ann Arbor, MI, USA). Events were gated as depicted in Figure 2. Initially, cells were
discriminated based on SSC-H and SSC-A as a multiplet cell exclusion criteria. Monocytes were
then differentiated into classical, intermediate and non-classical monocytes initially by FSC/SSC
characteristics and secondarily by CD14 and CD16 staining characteristics (16), with CD66b
exclusion (15). CD11b expression on each subset was then determined. Monocyte subsets are
presented as a percent of the total monocyte population, while CD11b expression is depicted as a
fold change from PRE to account for potential non-specific binding.
[Place Figure 2 Approximately Here]
Statistical Analysis
Changes in markers of muscle damage, circulating and intramuscular cytokines, as well
as monocyte subset distributions and CD11b expression, were analyzed by a two-way, between
subjects, repeated measures analysis of variance (ANOVA). In the event of a significant F ratio,
a one-way, within-subjects, repeated measures ANOVA for each group and a one-way, between-
subjects ANOVA at each time point with LSD pairwise comparisons were used for post-hoc
analysis. Significant time and group effects were subsequently analyzed with LSD pairwise
comparisons. Furthermore, given the impact of muscle biopsies on the inflammatory process (6),
intramuscular MCP-1 was analyzed as one-way repeated measures ANOVA for each group,
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similar to Della Gatta et al. (8). Data that were not normally distributed (according to the
Shapiro-Wilk test) underwent a natural log (LN) transformation. The area under the curve
(AUC) was also calculated for changes in circulating cytokines and myoglobin response from
PRE to 5H using a standard trapezoidal technique, and a one-way ANOVA was used to examine
differences among groups. Raw concentrations from PRE, IP, 1H and 5H were used to calculate
AUC prior to LN transformation. Additionally, interpretations of effect sizes (ηp2) were based on
those used by Wells and colleagues (23) for a similar study design: small effect (ηp2 < 0.06),
medium effect (ηp2 < 0.14) and large effect (ηp2 > 0.14). Pearson Product Moment Correlations
between markers of muscle damage and monocyte chemoattractants (MCP-1 and CX3CL1),
monocyte sub-populations and CD11b expression were assessed for all groups combined. As
changes in circulating CK usually do not manifest until 24-48 hours following resistance
exercise (6), markers of muscle damage included CK at 24H and 48H, as well as myoglobin area
under the curve (AUC). Significance was accepted at an alpha level of p ≤ 0.05 and all data are
reported as mean ± SD of the original, non-transformed data.
RESULTS
Participant Characteristics
No differences were observed between groups for age, height, body mass, BMI, maximal
strength (squat and leg press 1-RM), or dietary intake of calories, carbohydrates, protein or fats
(6). Furthermore, PPB and PL were significantly greater than CON at IP, 1H and 5H for
circulating myoglobin, while circulating CK was significantly increased in PPB at 24H and 48H,
and at 24H in PL compared to CON (6).
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Circulating Cytokines
Changes in circulating MCP-1 are depicted in Figure 3. A significant interaction was
observed for MCP-1 (Figure 3a; F = 3.23; p = 0.005; ηp2 = 0.16). Post hoc analysis indicated
significantly greater MCP-1 concentrations at 5H in PPB (p = 0.001) and PL (p = 0.012) than
CON. Significant increases in PPB from PRE at IP (p= 0.005), 1H (p = 0.010), and 5H (p <
0.001), as well as in PL from PRE to IP (p = 0.006) and 5H (p < 0.001), were also observed,
while significant reductions in MCP-1 were noted from PRE to 48H in both PPB (p = 0.048) and
PL (p = 0.010). AUC analysis indicated a significant interaction (Figure 3b; F = 3.53; p =
0.040), with a significantly greater MCP-1 response in PPB than CON (p = 0.014), and a trend
toward a significant increase between PPB and PL (p = 0.091).
No significant interaction was observed for CX3CL1 (F = 1.13; p = 0.345; ηp2 = 0.05),
though a significant effect of time was observed (F = 16.95; p < 0.001; ηp2 = 0.34) for all groups
combined. Pairwise comparisons indicated CX3CL1 was significantly elevated at IP (184.7 ±
70.9 pg∙ml-1) and 1H (171.9 ± 73.8 pg∙ml-1) compared to PRE (149.1 ± 84.9 pg∙ml-1; p < 0.001, p
= 0.004, respectively), 24H (138.9 ± 74.3 pg∙ml-1; p < 0.001) and 48H (130.5 ± 66.4 pg∙ml-1; p <
0.001), while IP was significantly greater than 5H (165.2 ± 107.7 pg∙ml-1; p = 0.005). AUC
analysis indicated no significant interaction (F = 0.87; p = 0.429).
[Place Figure 3 Approximately Here]
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Intramuscular Cytokine Protein Content
Changes in intramuscular MCP-1 protein content are depicted in Figure 3c. No
significant interaction was observed for intramuscular MCP-1 (F = 0.92; p = 0.473; ηp2 = 0.06),
however, due to the nature of this data, each condition was examined individually as previously
reported (8). Intramuscular MCP-1 content increased over time for each group individually
(CON: F = 12.29; p < 0.001; ηp2 = 0.67; PPB: F = 0.96; p < 0.001; ηp2 = 0.82; PL: F = 0.96; p <
0.001; ηp2 = 0.85). Intramuscular MCP-1 content increased in all groups compared to PRE (p <
0.05) however, only PPB increased from 1H to 5H (p = 0.028). Furthermore, intramuscular
MCP-1 content was higher at 5H than 48H for both PPB (p = 0.003) and PL (p = 0.016).
Monocyte Subset Distributions
Changes in monocyte subset distributions are depicted in Figure 4. A significant
interaction was observed for the proportion of classical monocytes (F = 27.99; p < 0.001; ηp2 =
0.47). Post hoc analysis indicated significantly reduced proportions of classical monocytes at IP
in PPB (p = 0.008) and PL (p = 0.003) compared to CON, while significant increases in the
proportion of classical monocytes at 1H were observed in PPB (p = 0.002) and PL (p = 0.006)
compared to CON.
A significant interaction was observed for the proportion of intermediate monocytes (F =
7.76; p < 0.001; ηp2 = 0.33). Post hoc analysis indicated significantly increased proportions of
intermediate monocytes at IP in PPB (p = 0.034) and PL (p = 0.001) when compared to CON,
while significantly reduced proportions were observed at 1H in PPB (p = 0.003) and PL (p =
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0.008) when compared to CON. Furthermore, there was a greater proportion of intermediate
monocytes in PPB than CON at 24H (p = 0.016) and 48H (p = 0.007).
A significant interaction was also observed for the proportion of nonclassical monocytes
(F = 7.43; p < 0.001; ηp2 = 0.32). Post hoc analysis indicated there was a significantly greater
proportion of nonclassical monocytes in PPB (p = 0.014) and PL (p = 0.015) than CON at IP.
[Place Figure 4 Approximately Here]
Monocyte CD11b Expression
Significant differences were observed between groups at PRE for CD11b expression on
intermediate monocytes (F = 3.59; p = 0.039) and nonclassical monocytes (F = 7.94; p = 0.002).
As such, analyses between groups for expression of CD11b on monocyte subsets were analyzed
as the percent of resting values (PRE representing 100%). Changes in CD11b expression on
monocyte subsets are depicted in Figure 5.
No significant interaction was observed for the change in CD11b expression on classical
monocytes (F = 1.79; p = 0.111; ηp2 = 0.10), however, a significant effect of time was identified
(F = 9.66; p < 0.001; ηp2 = 0.23) with all groups combined. Pairwise comparisons indicated a
significant increase from PRE to 1H (p < 0.001), 5H (p = 0.033) and 24H (p = 0.004).
Furthermore, changes in CD11b expression from PRE to 1H were significantly greater than
changes from PRE to IP (p = 0.001), 5H (p = 0.004), 24H (p < 0.001), and 48H (p < 0.001),
while the change from PRE to 24H was significantly greater than PRE to 48H (p = 0.002).
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No significant interaction was observed for the change in expression of CD11b on
intermediate monocytes (F = 1.86, p = 0.083, ηp2 = 0.10), however a significant main effect of
time was observed (F = 5.67, p = 0.001, ηp2 = 0.15) for all groups combined. Pairwise
comparisons indicated a significant increase in CD11b expression from PRE to 1H (p < 0.001).
No significant interaction (F = 1.92, p = 0.084, ηp2 = 0.11), nor main effect of time (F =
2.10, p = 0.104, ηp2 = 0.06) was observed for the change in expression of CD11b on non-
classical monocytes; however, a significant main effect for group (F = 4.41, p = 0.020, ηp2 =
0.22) was observed. Pairwise comparisons indicated that the average change in expression of
CD11b from PRE to all time points was lower in CON compared to PPB (p = 0.006). No
difference was observed between PL and CON (p = 0.121) or PPB (p = 0.106).
[Insert Figure 5 approximately here]
Correlations
Significant correlations were observed between circulating markers of muscle damage
and monocyte chemoattractants. Circulating MCP-1 concentrations at 5H and 24H were
significantly correlated with CK concentrations at 24H (r = 0.406, p = 0.014; r = 0.505, p =
0.002, respectively), and 48H (r = 0.396, p = 0.017; r = 0.491, p = 0.002, respectively). MCP-1
concentration at 24H was also significantly correlated with myoglobin AUC (r = 0.435; p =
0.007).
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CX3CL1 concentrations at 5H and 24H were correlated with circulating CK
concentration at 24H (r = 0.433, p = 0.011; r = 0.408, p = 0.017, respectively) and 48H (r =
0.394, p = 0.021; r = 0.364, p = 0.034, respectively). CX3CL1 concentrations were also
correlated with myoglobin AUC at 1H (r = 0.340, p = 0.046), 5H (r = 0.503, p = 0.002) and 24H
(r = 0.434, p = 0.009).
Significant correlations were observed between markers of muscle damage and changes
in CD11b expression on monocyte subsets. CK concentrations at 24H were significantly
correlated with the change in CD11b expression on intermediate monocytes at IP (r = 0.439, p =
0.001), and nonclassical monocytes at IP (r = 0.413, p = 0.017), 1H (r = 0.392, p = 0.024) and
5H (r = 0.374, p = 0.032). Furthermore, CK concentrations at 48H were significantly correlated
with the change in CD11b expression at IP on classical monocytes (r = 0.365, p = 0.037), and
intermediate monocytes (r = 0.452, p = 0.008). Additionally, the change in CD11b expression
on nonclassical monocytes was correlated at IP (r = 0.450, p = 0.009), 1H (r = 0.402, p = 0.020),
5H (r = 0.345, p = 0.049), 24H (r = 0.446, p = 0.009) and 48H (r = 0.495, p = 0.003). No
significant correlations were observed between the change of CD11b expression on leukocytes
and myoglobin AUC. Correlations with leukocyte subset proportions are displayed in Table 1.
[Place Table 1 Approximately Here]
DISCUSSION
The primary findings of this investigation were that resistance exercise elicited
significant recruitment, and mobilization of monocytes. As expected, resistance exercise
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stimulated an increase of circulating MCP-1 concentrations immediately following exercise and
a further increase at 5H. While the time course of this response does not appear to be influenced
by polyphenol supplementation, PPB did have a significantly greater AUC response than CON,
while PL did not. Additionally, resistance exercise induced significant mobilization of
intermediate and nonclassical monocyte subtypes immediately following resistance exercise,
followed by a supercompensation of the classical subset, while the PPB group experienced an
elevated proportion of intermediate monocytes 48 hours into recovery. Furthermore, classical
and intermediate monocytes increased expression of CD11b at 1H, while only classical
monocytes maintained this elevated expression for 24 hours. It is important to note, however,
this increase may have been due to the effect of the muscle biopsy, as this increase was noticed
for all groups combined. Interestingly, however, while polyphenol supplementation may have
enhanced the CD11b response to exercise compared to CON on nonclassical monocytes, though,
PPB supplementation may have suppressed CD11b expression on intermediate and nonclassical
monocytes at rest.
Skeletal muscle damage produces a robust immune response, characterized by an
increased accumulation of phagocytic cells within the damaged tissue (10). MCP-1 plays an
integral role in the acute immune response by serving as the primary chemoattractant for
classical monocytes (16,21). Our results indicated that the response of circulating MCP-1 to
resistance exercise may be biphasic; characterized by an initial increase immediately following
exercise, and a second, larger increase at 5H. To the best of our knowledge, only two
investigations have examined the acute response of circulating MCP-1 following dynamic
resistance exercise (23,24). The immediate increase of circulating MCP-1 observed in this study
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is in contrast to the decrease in MCP-1 concentrations reported 30-min following resistance
exercise in untrained men (24). It is similar, however, to the immediate increase reported
following a lower body resistance exercise protocol in resistance trained men (23). While
Ihalainen et al. (24) utilized a similar population as this study (e.g., previously untrained men),
the exercise stimulus only used multiple sets of a single leg press exercise. In contrast, the
current study required participants to exercise with multiple lower body exercises, similar to
Wells and colleagues (23). The greater number of repetitions (140 total repetitions compared to
50 in the Ihalainen et al. study), and exercises (Squat, Leg Press and Leg Extension compared to
Leg Press only) performed in this study may have resulted in a greater muscle damage (6,24).
Given the observed correlations between markers of muscle damage and MCP-1 in the current
study, the observed increase in MCP-1 concentration may be influenced by muscle damage or
training volume, and may be independent of training status.
The biphasic MCP-1 response observed was not found in previous studies examining the
MCP-1 response to resistance exercise (23,24). Investigations utilizing protocols designed to
elicit muscle damage, however, have seen a secondary response approximately 5-hr into
recovery (1). The participants in those studies had a similar training background as those
recruited in this present study. Therefore, in addition to training volume, the biphasic response
of MCP-1 may also be a function of novel muscle action.
Within the muscle, MCP-1 was found to increase from PRE in all conditions, and at all
time points. Exercise appeared to increase intramuscular MCP-1, and PPB supplementation may
delay this response. Previous investigations have demonstrated increased intramuscular MCP-1
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in response to both aerobic (32), and resistance exercise (8). Our results are similar to the
findings of Della Gatta and colleagues (8), who demonstrated increases at two and four hours
following resistance exercise. Increases in intramuscular MCP-1 were also observed in the CON
group, however, indicating a potential influence of the muscle biopsy on the MCP-1 response
(6). Therefore, it is difficult to isolate the effect of resistance exercise from that of the biopsy.
Regardless of the source, the increases observed in circulating and intramuscular MCP-1 likely
recruit classical monocytes to the site of tissue damage (21).
While the recruitment of classical monocytes is governed by MCP-1, the recruitment of
other monocyte subsets is governed primarily by CX3CL1 (22). Circulating CX3CL1
concentrations increased at both IP and 1H for all groups combined. Others have reported
significant increases in CX3CL1 mRNA two hours post-resistance exercise (8), while Della
Gatta and colleagues (8) demonstrated a return to baseline levels at 4-hr post-exercise. To our
knowledge, only one investigation has examined circulating CX3CL1 concentrations after
exercise, and has reported increases for two hours following unilateral cycling (33). As this
investigation (33) did not have a control group, it is difficult to distinguish between the effects of
the exercise and that of the biopsy. Nonetheless, as CX3CL1 is synthesized by endothelial cells
(34), it will remain bound to the endothelial surface unless cleaved by TNF-α and IL-1β (35). As
TNF-α and IL-1β are pro-inflammatory cytokines (7), the observed increase in CX3CL1 may be
more indicative of a pro-inflammatory environment, which will be observed after resistance
exercise, or potentially following skeletal muscle biopsies (6,36).
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As with markers of monocyte recruitment, expansion of the monocyte population has
been well documented (9); however, the mobilization of specific subtypes is less defined.
Previously, Wells and colleagues (19) have demonstrated no change in classical monocytes
immediately following resistance exercise, with significant increases from 1-5 hours into
recovery in resistance-trained men (19). While to our knowledge, this is the first investigation to
document changes in circulating intermediate and nonclassical monocyte proportions following
resistance exercise, previous studies, using an aerobic exercise model, have demonstrated
significant mobilization of the nonclassical subset immediately following exercise (17,18). This
selective mobilization of CD16+ monocytes may be mediated by catecholamines (37), though
exercise intensity appears to drive the response (39). Increases in both the intermediate and
nonclassical monocyte populations have also been demonstrated following aerobic exercise,
though this was demonstrated while using a mixed-gender population (17). Considering
nonclassical monocytes appear to respond differently to exercise in women compared to men
(39), the increased intermediate monocyte proportion observed by Booth et al. (17) may be
driven by the sex of the participants. As this investigation utilized only men, the increased
intermediate monocytes observed may be specific to resistance exercise.
Further increases in intermediate monocytes at 24H and 48H in this investigation
appeared to occur at the expense of the classical monocyte subset, with no change in the
nonclassical proportion. Furthermore, PPB demonstrated a significantly greater intermediate
monocyte population than CON at both these time points. While data following ischemic tissue
damage has shown elevations in intermediate monocytes 24- and 48-hours post injury (20), to
our knowledge, no investigations have examined the monocyte subset response to exercise 24-
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and 48-hours into recovery. Furthermore, Tapp and colleagues (20) have reported that changes
in intermediate monocytes were associated with the extent of tissue damage. This is consistent
with the correlations observed in this study (r = 0.380 to 0.452) between markers of muscle
damage and the intermediate monocyte proportions at 24H and 48H. Given that this resistance
exercise bout resulted in a greater increase in CK in PPB versus PL (6), it is possible the
observed increase in intermediate monocytes at 24H and 48H is a function of muscle damage as
opposed to the effects of polyphenol supplementation. Therefore, it is possible that intermediate
monocytes increase in proportion to the magnitude of skeletal muscle damage that occurs post-
exercise, though future studies must confirm this.
The involvement of CD11b in the transendothelial migration process makes CD11b a key
regulator of the phagocyte migration to damaged tissue (26). Previous reports have
demonstrated elevated CD11b expression on classical monocytes immediately following and 1-
hour post-resistance exercise (23), while another demonstrated significant increases 30-min
following a resistance exercise bout (25). The disparity in the time course reported between
these studies may be due to the methods used to identify monocytes, and the lack of
differentiation between classical and intermediate monocytes (25). Consequently, Wells et al.
(23) suggested that the response of CD14++ monocytes may have been influenced by
intermediate monocytes. The delayed increase of intermediate monocytes in this investigation
support the assertion by Wells and colleagues (23); however, the current study also observed a
delayed increase (at 1H) of CD11b on classical monocytes. This difference may be due to the
lower level of training of the participants in our study, however, the methods of analysis may
have also played a role, as we examined changes from PRE, as opposed to raw MFI expressions.
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Polyphenol supplementation (PPB) for 28 days also appeared to reduce CD11b
expression on intermediate and nonclassical monocytes at rest compared to CON and PL.
CD11b expression on monocytes was not examined prior to the onset of supplementation,
therefore we can only speculate in relating this decreased expression of CD11b to polyphenol
supplementation. However, there does appear to be support for this from an in vitro model,
which demonstrated a significant downregulation of CD11b, as well as reduced chemotaxis and
adherence in response to incubation with polyphenols (28). However, others have suggested that
decreased CD11b expression on monocytes may have beneficial health benefits, as polyphenol
associated decreases in CD11b expression on monocytes has been previously reported to have
potential beneficial effects in cardiovascular disease (27).
In this study, no differences in the time course of CD11b expression were observed
between groups for classical or intermediate monocytes. However, participants in PPB
demonstrated significantly greater increases in CD11b expression on nonclassical monocytes
during 48-hr of recovery than CON. While the expression of CD11b on nonclassical monocytes
is reduced compared to other subsets (40), it is unclear why this difference occurred.
Nonetheless, the role of CD11b in transendothelial migration (26), and the propensity of
nonclassical monocytes to polarize to M2 macrophages (41) may indicate an increased tendency
to transmigrate, that would likely have no deleterious effects on recovery. Therefore, polyphenol
supplementation may reduce monocyte adherence and chemotaxis at rest, however, may only
have a limited effect on the exercise response, though a greater understanding of how other
components to the cell adhesion cascade respond to polyphenol supplementation is needed.
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Together, the results of this investigation demonstrate that resistance exercise initiates
monocyte recruitment, and mobilization. Furthermore, polyphenol supplementation appears to
modestly influence the migration of monocytes, reducing CD11b expression at rest, and possibly
enhancing expression on nonclassical monocytes following exercise. Therefore, this may
modify the transmigration of monocytes into the tissue, although examinations that confirm this
within muscle are needed. Unfortunately, the muscle biopsies used in this investigation appear to
have influenced some of the results of this study, evidenced by the increased intramuscular
MCP-1 and circulating monocyte CD11b expression in CON. While this has been demonstrated
to occur previously (6), the biopsies were vital to the acquisition of this data. Although we
utilized microbiopsies, as opposed to percutaneous biopsies with suction (8) to reduce the total
quantity of tissue collected, this also increases the ratio of tissue exposed to the trauma of the
biopsy procedure itself, in relation to the total volume of tissue collected, potentially inflating the
MCP-1 concentration of our sample. Furthermore, as this investigation did not use absolute cell
counts, we are unable to indicate absolute changes in monocyte subset populations.
Additionally, as CD16 is a marker for NK cells (42), the CD16+ nonclassical monocyte gate may
have contained a small number of NK cells. Future research should examine the changes in
monocyte subset population counts following resistance exercise. Furthermore, these
investigations should examine the expression of various chemotactic receptors, namely CX3CR1
and CCR2, following resistance exercise. Finally, given that polyphenol supplementation
appears to influence the expression of cell adhesion molecules, future studies should examine
earlier components of the cell adhesion cascade to provide a greater understanding of the
propensity for monocytes to undergo transendothelial migration.
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ACKNOWLEDGMENTS
This study was funded by Kemin Foods, L.C. (Des Moines, IA). The authors would like to thank
Michael J. Redd, Michael B. La Monica, Carleigh H. Boone, Kayla M. Baker, Joshua J. Riffe,
Tyler W.D. Muddle, and Ran Wang for their assistance with data collection for this manuscript.
The authors would also like to thank Frank Zaldivar, PhD and Fadia Haddad, PhD for their
assistance with the flow cytometry protocols and data acquisition. This investigation is
registered on clinicaltrials.gov with reference number NCT02442245.
CONFLICT OF INTEREST
Kelli A. Herrlinger is an employee of Kemin Foods, L.C. All other authors have no actual or
potential conflicts of interest to report. As such, the results of this study are presented clearly,
honestly, and without fabrication, falsification, or inappropriate data manipulation. The results
of this study do not constitute endorsement by the American College of Sports Medicine.
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FIGURES AND TABLES
Figure 1: Study Design. At least 72 hours prior to testing, participants completed their 1-RM
testing of the squat, leg press and leg extension. During the acute exercise protocol, participants
provided blood samples prior to exercise (PRE), immediately (IP), one- (1H), five- (5H), 24-
(24H) and 48- (48H) hours following exercise. Muscle biopsies were obtained at PRE, 1H, 5H
and 48H.
Figure 2: Identification of Monocyte Subsets.
Cells were initially gated based on side scatter – area (SSC-A) and height (SSC-H) for exclusion
of mutiplet cells (A). Monocytes were then gated based on forward scatter (FSC) and SSC
characteristics (B), isolated as CD66b- (C), before final discrimination, based on CD14 and
CD16 expression (D). Expression of CD11b for each subset was then assessed.
Figure 3: Macrophage Chemoattractant Protein 1 (MCP-1) response to Resistance
Exercise.
Data from circulating MCP-1 concentration (expressed in A) are presented from polyphenol
(PPB; n = 13), placebo (PL; n = 15) and control (CON; n = 10) groups at pre-exercise (PRE), as
well as immediately (IP), one- (1H), five- (5H), 24- (24H), and 48- (48H) hours following
exercise. Area under the curve (AUC) was calculated from circulating concentrations from PRE
to 5H (expressed in B). Data reflecting intramuscular MCP-1 protein content (expressed in C)
was analyzed from a subset of the total population (PPB: n = 11; PL: n = 12; CON: n = 7).
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* Indicates a significant difference compared to corresponding PRE (p < 0.05)
^ Indicates a significant difference with CON (p < 0.05)
# Indicates a significant differences compared to corresponding 5H (p < 0.05)
Figure 4: Monocyte Subset Proportions following Resistance Exercise
Supplement (PPB; n= 9), placebo (PL; n= 10) and control (CON; n= 15) groups were analyzed
for changes in the monocyte subset proportions pre exercise (PRE), as well as immediately (IP),
one- (1H), five- (5H), 24- (24H), and 48- (48H) hours post exercise.
* Significantly different than corresponding value for PRE (p < 0.05)
^ Significantly different than corresponding value for CON (p < 0.05)
Figure 5. CD11b Expression on Monocyte Subsets.
Changes in CD11b Expression on classical (A), intermediate (B) and nonclassical (C) monocytes
were assessed between polyphenol (PPB; n = 11), placebo (PL; n = 15) and control (CON; n = 9)
groups. Changes were assess from pre-exercise (PRE) to immediately (IP), one- (1H), five- (5H),
24- (24H) and 48- (48H) hours after exercise.
* Significant Change from PRE for all groups combined (p < 0.05).
^ Significant Main Effect for Group compared to CON (p < 0.05).
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Figure 1
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Figure 2
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Figure 3
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Figure 4
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Figure 5
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Table 1: Correlations between Monocyte Subset Proportions and Markers of Muscle
Damage.
Myoglobin AUC
24H CK Concentration
48H CK Concentration
CLASS
INTER
NC
CLASS
INTER
NC
CLASS
INTER
NC
IP
r
-0.664
-
0.775
-0.692
-
0.719
-0.583
0.399
0.555
p
<0.001
<0.001
<0.001
<0.001
<0.001
0.024
0.001
1H
r
-0.359
-
0.535
-
-
-
-
-
-
p
0.040
0.001
5H
r
-0.548
-
0.568
-0.361
-
-
-
-
-
p
0.001
0.001
0.042
24H
r
-0.543
-
0.609
-0.466
-
0.434
-0.434
0.380
-
p
0.001
<0.001
0.007
0.013
0.013
0.032
48H
r
-0.640
0.412
0.631
-0.565
0.452
0.471
-0.457
0.387
0.361
p
<0.001
0.017
<0.001
0.001
0.009
0.006
0.009
0.029
0.042
Pearson Product Moment Correlation coefficients between markers of muscle damage and
monocyte subset proportion for all groups combined. Correlation coefficient (r) and significance
(p) are presented, while non-significant correlations are represented with a “-”. Data were
analyzed on classical (CLASS), intermediate (INTER) and nonclassical monocytes (NC).
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