Physical Exercise Reduces the Expression of RANTES and Its CCR5 Receptor in the Adipose Tissue of Obese Humans

Research ArticlePhysical Exercise Reduces the Expression of RANTES andIts CCR5 Receptor in the Adipose Tissue of Obese Humans

Engin Baturcam12 Jehad Abubaker1 Ali Tiss1 Mohamed Abu-Farha1

Abdelkrim Khadir1 Fahad Al-Ghimlas3 Irina Al-Khairi1 Preethi Cherian1 Naser Elkum4

Maha Hammad1 Jeena John1 Sina Kavalakatt1 Cynthia Lehe1 Samia Warsame1

Kazem Behbehani134 Said Dermime5 and Mohammed Dehbi16

1 Department of Biomedical Research Dasman Diabetes Institute 1180 Dasman Kuwait2 Queensland Childrenrsquos Medical Research Institute University of Queensland Brisbane QLD 4029 Australia3 Fitness and Rehabilitation Centre Dasman Diabetes Institute 1180 Dasman Kuwait4Department of Biostatistics and Epidemiology Dasman Diabetes Institute 1180 Dasman Kuwait5 King Fahad Specialist Hospital Dammam 15215 Saudi Arabia6Diabetes Research Center Qatar Biomedical Research Institute Education City PO Box 5825 Doha Qatar

Correspondence should be addressed to Mohammed Dehbi mdehbiqforgqa

Received 19 December 2013 Accepted 30 March 2014 Published 17 April 2014

Academic Editor Chiara De Luca

Copyright copy 2014 Engin Baturcam et alThis is an open access article distributed under theCreativeCommonsAttribution Licensewhich permits unrestricted use distribution and reproduction in any medium provided the original work is properly cited

RANTES and its CCR5 receptor trigger inflammation and its progression to insulin resistance in obese In the present study weinvestigated for the first time the effect of physical exercise on the expression of RANTES and CCR5 in obese humans Fifty-seven adult nondiabetic subjects (17 lean and 40 obese) were enrolled in a 3-month supervised physical exercise RANTES andCCR5 expressions were measured in PBMCs and subcutaneous adipose tissue before and after exercise Circulating plasma levelsof RANTES were also investigatedThere was a significant increase in RANTES and CCR5 expression in the subcutaneous adiposetissue of obese compared to lean In PBMCs however while the levels of RANTES mRNA and protein were comparable betweenboth groups CCR5 mRNA was downregulated in obese subjects (119875 lt 005) Physical exercise significantly reduced the expressionof both RANTES and CCR5 (119875 lt 005) in the adipose tissue of obese individuals with a concomitant decrease in the levels ofthe inflammatory markers TNF-120572 IL-6 and P-JNK Circulating RANTES correlated negatively with anti-inflammatory IL-1ra(119875 = 0001) and positively with proinflammatory IP-10 and TBARS levels (119875 lt 005) Therefore physical exercise may provide aneffective approach for combating the deleterious effects associated with obesity through RANTES signaling in the adipose tissue

1 Introduction

Chronic low-grade inflammation and aberrant regulation ofthe stress response system are two prominent hallmarks ofobesity a major risk factor for the development of insulinresistance type 2 diabetes metabolic syndrome and car-diovascular diseases [1 2] Sedentary lifestyles and excessivefood intake are considered as key contributors to this chroniccondition The white adipose tissue has been identified asthe predominant site of obesity-associated inflammatoryreactions due to its infiltration by immune inflammatory cells

such as monocytes macrophages Th1 T cells and dendriticcells [2ndash4] These immune cells together with adipocytesand stromal vascular cells constitute a cellular network thatproduces various inflammatory mediators Obesity-inducedinflammatory response impairs insulin signaling in insulin-responsive organs and causes systemic insulin resistancewhich leads to a perturbation of glucose homeostasis andultimately type-2 diabetes [5 6] Studies on mice indicatedthat obesity also alters the balance between pro- and anti-inflammatory activities in adipose tissue by promoting thephenotypic switch fromM2 anti-inflammatory macrophages

Hindawi Publishing CorporationMediators of InflammationVolume 2014 Article ID 627150 13 pageshttpdxdoiorg1011552014627150

2 Mediators of Inflammation

to M1 proinflammatory macrophages and thereby perpetuat-ing further the inflammatory response and insulin resistance[5 7]

Regulated upon Activation Normal T cells Expressed andSecreted (RANTES or CCL5) is a powerful proinflamma-tory mediator of the CC chemokine family that regulatesthe mobilization and in certain cases promotes survivalof immune inflammatory cells from the bloodstream intotissues and other areas of injury and infection [3 8 9]Although the chemotactic activity of RANTES on immunecells to injured and infected areas is beneficial sustainedproduction of RANTES is associatedwith several detrimentaleffects such as atherosclerosis [10 11] arthritis rheumatoid[12] liver disease [13 14] and viral infection [15] that sharein common chronic inflammatory response Consistent withits critical role in the pathophysiology of these chronicinflammatory-related diseases treatments that interfere withRANTES signaling such as neutralizing antibody [16 17]peptide mimetics [18 19] and pharmacological inhibitors[20 21] are associated with improved outcomes RANTESorchestrates its effects through binding to one of its cog-nate receptors including CCR1 CCR3 and CCR5 [22 23]The crucial role of CCR5 in mediating the inflammatoryresponse in adipose tissue was demonstrated recently byusing CCR5 knockout mice that showed a dominant shiftfrom M1-phenotype to M2-phenotype which contributed toattenuated inflammatory response and improved impairedglucose tolerance and insulin sensitivity in response to diet-induced obesity [24]

Physical exercise is an important component of healthylifestyle that is widely advocated as a first line for thetreatment and management of obesity insulin resistancediabetes and cardiovascular diseases [25ndash27] Previous stud-ies linked the protective effect of physical exercise to theimprovement of the inflammatory and stress responses [28ndash30] The mechanisms by which physical exercise mediatesits anti-inflammatory effect are multiples and they include areduction of visceral fat mass with subsequent decrease in theproduction of proinflammatory adipokines and a reductionin the circulating number of proinflammatory monocytes infavor of an increase in the circulating number of regulatory Tcells [31 32] Other studies revealed also that physical exerciseprevents monocytes and macrophages from infiltrating intoadipose tissue and induces a phenotypic switching fromM1-phenotype macrophages to M2-macrophages within theadipose tissue [33] Other beneficial effects of physical exer-cise include attenuated activation of c-Jun NH

2-terminal

kinase [34] together with improvement of lipid glucose andoxidative homeostasis [35ndash37]

In the current study we investigated the effect of a3-month physical exercise on the expression and releaseof RANTES in obese subjects Given the critical role ofRANTES and its CCR5 receptor in promoting obesity-inducedmetabolic inflammation we hypothesized that phys-ical exercise may mediates its anti-inflammatory effect inpart by impairing RANTES signaling pathway via downreg-ulation of RANTES andor its CCR5 receptorWe report herefor the first time a decrease in the expression of bothRANTES

and CCR5 receptor by physical exercise in the adipose tissueof obese humans

2 Materials and Methods

21 Study Population Thestudywas conducted on adultmaleand female nondiabetic subjects consisting of 17 lean (BMI= 20ndash249 kgm2) and 40 obese (BMI = 30ndash40 kgm2) Allsubjects gave written informed consent and the study wasapproved by the Ethical Review Board of Dasman DiabetesInstitute (Reference number RA-2010-003)

Participants that followed any physical exercise withinthe last 6 months prior to this study as well as those withprior major illness or intake of medications or supplementsknown to influence the body composition or bone masswere excluded from the study None of the participants thatwere enrolled in this study reported any cerebrovascularcomplication kidney disease asthma and food and drugallergies and chronic viral infection According to themedicalquestionnaire none of the participants was under low-calorieor special diet Furthermore no specific diet or nutritionalchange was prescribed to our subject during the 3 months ofphysical exercise protocol as our aim was mainly to investi-gate the sole effect of physical exercise on those subjects

22 Exercise Protocol All eligible subjects were enrolled to asupervised exercise program at the Fitness and RehabilitationCenter (FRC) of Dasman Diabetes Institute Prior to exerciseprescription each individual underwent a symptom-limitedmaximal incremental cardiopulmonary exercise test as wellas a muscle strength and endurance test The details of thesetests and the exercise protocol have been previously described[38] It consisted of a combination of bothmoderate intensityof aerobic exercise and resistance training using either tread-mill or cycling Each exercise session included 10 minutes ofwarming-up and cooling down steps at 50ndash60 of max HRalong with 40 minutes of the prescribed exercise program at65ndash80 of max HR For the duration of the 3-month periodparticipants exercised 3 to 5 times per week and they wereinstructed to reach and maintain the recommended heartrate range This was achieved by regular monitoring of theheart rate during the aerobic training Strength training wasperformed 2 to 3 times a week according to the programplan Exercise intensity duration and blood pressures wererecorded for each session All trainings were supervisedby qualified fitness professionals from FRC To assess theeffectiveness of the exercise the same physical stress andfitness tests were performed for all subjects at the end of theexercise program

23 Blood and Tissue Sampling Venous peripheral bloodand subcutaneous adipose tissue biopsies were obtainedprior to the start of the 3-month protocol and within 2to 3 days after its completion PBMCs were prepared fromblood using Ficoll-Hypaque density gradient centrifugationmethod Plasma and serum were prepared using vacutainertubes and then aliquoted and stored at minus80∘C Subcutaneoussuperficial adipose tissue biopsies (sim05 g) were obtainedfrom the periumbilical area by surgical biopsy after a local

Mediators of Inflammation 3

anesthesia Once removed the biopsy was rinsed in cold PBSdivided into 4 pieces and stored appropriately until assayed

24 Anthropometric Measurements and Blood BiochemistryAnthropometric measurements were taken at the baselineand after 3months of exercise Glucose and lipid profiles weremeasured on the Siemens Dimension RXL chemistry ana-lyzer (Diamond Diagnostics Holliston MA) HemoglobinA1c (HBA1c) was determined using the Variant device(BioRad Hercules CA) Plasma levels of inflammatory andmetabolic markers were measured using the Bio-plex 27-plex human cytokine and 12 Bio-plex human diabetes kitsrespectively (BioRad Hercules CA) Data were analyzedwith Bio-plexManager software version 6 (BioRad HerculesCA) Lipid peroxidation was assessed by measuring plasmalevels of malonaldehyde using TBARs Assay Kit (CaymanChemical Company Ann Arbor MI) Serum levels of ROSwere determined using the OxiSelect ROS Assay Kit (CellBiolabs Inc SanDiego CA) All the above assays were carriedout according to the instructions of the manufacturers

25 Quantitative Real-Time PCR (qRT-PCR) Total RNA wasextracted from frozen adipose tissue and PBMCs usingRNeasy Lipid Tissue Mini Kit and AllPrep RNAProtein Kitrespectively (Qiagen Inc Valencia CA) The cDNA wassynthesized from total RNA sample using High CapacitycDNA Reverse Transcription Kits (Applied Biosystems Fos-ter City CA) qRT-PCR was performed on Rotor-Disc 100system using SYBR Green normalized to Gapdh (QiagenInc Valencia CA) PCR primer used were as followsRANTES For 51015840-TTTGCCTACATTGCCCGC-31015840 RANTESRev 51015840-TTTCGGGTGACAAAGACGACT-31015840 CCR5 For51015840-CAAAAAGAAGGTCTTCATTACACC-31015840 and Ccr5Rev 51015840-CCTGTGCCTCTTCTTCTCATTTCG-31015840 TNF-120572For 51015840-AGAGGGAAGAGTTCCCCAGG-31015840 TNF-120572 Rev51015840-ATTGGCCAGGAGGGCATT-31015840 IL-6 For 51015840-AGA-AAGGAGAGTCACAGGTGAGC-31015840 IL-6 Rev 51015840-TGT-CTGGGAAAGAATACCAGAA-31015840 Gapdh For 51015840-AAC-TTTGGCATTGTGGAAGG-31015840 and Gapdh Rev 51015840-TGT-GAGGGAGATGCTCAGTG-31015840 Relative expression wasassessed by using the ΔΔCT method [39]

26 Western Blot Analysis Cell lysates were prepared fromPBMCs by the addition of RIPA buffer (50mM Tris-HCl pH75 150mMNaCl 1 Triton x100 1mMEDTA 05 Sodiumdeoxycholate and 01 SDS) Protein concentration wasdetermined by Bradfordmethod using globulin as a standardand 20120583g of proteins was resolved on 10 SDS-PAGE gelsProteinswere transferred onto PVDFmembranes and probedwith primary and secondary antibodies using standard pro-tocols Protein bands were visualized by chemiluminescenceand the images were captured by using the Versadoc 5000system (BioRad Hercules CA)The primary antibodies usedin this study are raised against RANTES (Abcam CambridgeMA) and Actin (Santa Cruz Biotechnology Santa Cruz CA)For densitometric analysis the intensity of the bands wasdetermined using Quantity One Software (BioRad HerculesCA)

27 Immunohistochemistry Formalin-fixed and paraffin-embedded adipose tissue sections were deparaffinizedand rehydrated prior to antigen retrieval by boiling in theunmasking solution (Dako Denmark) The endogenousperoxidase was quenched using 3 H

2O2for 60min at RT

Sections were blocked with 5 fat-free milk for 60min at RTfollowed by 1 BSA for another 60min and then incubated at4∘C for overnight with primary antibodies against RANTES(Abcam Cambridge MA) TNF-a (Abcam CambridgeMA) IL-6 (Novus Biologicals Littleton CO) and Phospho-JNK (Cell Signaling Technology Inc Danvers MA)Staining with horseradish conjugated secondary antibody(Dako Denmark) was done for 60minutes at RT Colors weredeveloped using DAB kit (Dako Denmark) and sectionswere counterstained with hematoxylin (Sigma Aldrich StLouis MO) Quantification of the immunohistochemicaldata was done using Aperio software version 63 (MolecularDevices Downingtown PA) with an established arbitrarythreshold

28 Flow Cytometry Analysis Approximately 5 times 105 offrozen PBMCs were thawed and washed with RPMI mediumsupplemented with 10 FBS antibiotics and L-glutamine(complete media) For RANTES staining cells were culturedovernight in complete media at 37∘C and then washed withPBS containing 2 FBS followed by FACS buffer (BD Bio-sciences San Jose CA) and then costained with anti-humanAPC-conjugated RANTES (RampD Systems) FITC-conjugatedCD3 (MCA463F AbD Serotec Raleigh NC) and PE-conjugated CD14 (FAB3832P RampD Systems MinneapolisMN) antibodies for 45minutes on ice Cells were thenwashedtwice with permeabilization buffer (eBioscience San DiegoCA) and resuspended in 300 120583L FACS buffer for analysisRegarding CCR5 staining cells were allowed to recover for1-2 hours in complete media The cells were then cultured incomplete media in presence of 2 120583Mmonensin (eBioscienceSan Diego CA) at 37∘C and 5 CO

2for 16 hours PBMCs

were thenwashedwith FB and surface stained simultaneouslywith FITC-conjugated CD3 (MCA463F) and PE-conjugatedCD14 (FAB3832P) antibodies After 45minutes of incubationon ice in the dark cells were washed twice and fixed withfixation buffer (eBioscience San Diego CA) for 20 minutesCells were then washed twice with permeabilization buffer(eBioscience San Diego CA) according to manufacturerrsquosrecommendation followed by staining with CCR5-APC anti-body (FAB1802A RampD Systems Minneapolis MN) for 20minutes in the dark Finally the cells were resuspendedin 300 120583L FACS Buffer for analysis Controls for specificlabeling were prepared with isotype-matched controls foreach sample Acquisition and analysis were carried out ona FACS Canto II flow cytometer using FACSDiva software(BD Biosciences San Jose CA) Typically 30000 events forPBMCS were acquired After forward scatter (FSC) and sidescatter (SSC) gating on either lymphocytes or monocytes theRANTES expression level was determined in the CD3 as wellas in CD14 subsets

29 Statistical Analysis Statistical analyses were performedwith SAS version 92 (SAS Institute Inc Cary NC) Unless

4 Mediators of Inflammation

Table 1 Physical clinical and biochemical characteristics of the study population at baseline

Lean (119899 = 17) Obese (119899 = 40) 119875 valuePhysical and clinical characteristicsGender (MF) 611 2218 018Age (year) 3724 plusmn 249 4345 plusmn 187 006BMI (kgm2) 2278 plusmn 055 3425 plusmn 048 lt00001PBF () 2586 plusmn 108 3867 plusmn 073 lt00001Waist (cm) 7604 plusmn 546 10753 plusmn 282 lt00001Hip (cm) 8408 plusmn 573 11515 plusmn 268 lt00001Resting HR (beatmin) 8217 plusmn 789 7865 plusmn 198 094SBP (mmHg) 11533 plusmn 437 12794 plusmn 312 009DBP (mmHg) 8000 plusmn 258 8159 plusmn 232 094119881O2 Max (mLkgmin) 2009 plusmn 118 1812 plusmn 117 022Metabolic markersCholesterol (mmolL) 497 plusmn 023 532 plusmn 015 020HDL (mmolL) 128 plusmn 006 114 plusmn 004 006LDL (mmolL) 322 plusmn 022 335 plusmn 014 062TG (mmolL) 103 plusmn 022 172 plusmn 014 0013Glucose (mmolL) 496 plusmn 018 543 plusmn 012 0035HBA1c () 553 plusmn 010 585 plusmn 007 0013C-peptide (ngmL) 267 plusmn 029 303 plusmn 018 029Insulin (ngmL) 259 plusmn 046 347 plusmn 029 012Leptin (ngmL) 471 plusmn 090 866 plusmn 058 00006PAI-1 (ngmL) 326 plusmn 038 364 plusmn 024 041Resistin (ngmL) 122 plusmn 011 094 plusmn 007 004Visfatin (ngmL) 978 plusmn 213 1023 plusmn 138 086Inflammatory markersTNF-120572 (pgmL) 2564 plusmn 377 2875 plusmn 213 049IL-1120573 (pgmL) 125 plusmn 019 133 plusmn 012 072IL-1ra (pgmL) 105 plusmn 11 927 plusmn 73 038IL-4 (pgmL) 230 plusmn 026 209 plusmn 017 050IL-6 (pgmL) 556 plusmn 056 499 plusmn 036 040IL-10 (pgmL) 229 plusmn 071 245 plusmn 045 085IP-10 (pgmL) 344 plusmn 50 558 plusmn 32 00008MCP-1 (pgmL) 932 plusmn 100 1003 plusmn 064 056MIP-1a (pgmL) 926 plusmn 332 341 plusmn 180 013MIP-1b (pgmL) 2242 plusmn 694 3019 plusmn 444 036RANTES (ngmL) 124 plusmn 018 175 plusmn 012 0023Oxidative stress markersROS (mM) 142 plusmn 006 151 plusmn 006 033TBARS (120583M) 122 plusmn 012 149 plusmn 008 007Data were adjusted for age and gender and presented as mean plusmn SE BMI body mass index PBF percent body fat HR heart rate SBP systolic blood pressureDBP diastolic blood pressure 119881O2 Max maximum oxygen consumption HDL high density lipoprotein LDL low density lipoprotein and TG triglycerides

otherwise stated all descriptive statistics for the variablesin the study were reported as means plusmn standard errorNonparametric Mann-Whitney test was used to determinesignificance of difference in means between the two groupsas indicated in the figure legends Correlations between vari-ables were calculated with the Spearmanrsquos rank correlationtest Differences were considered statistically significant at 119875values less than 005

3 Results

31 Baseline Characteristics of Study Population Thephysicalcharacteristics of the two groups namely lean (119899 = 17)and obese (119899 = 40) enrolled in this study are displayedin Table 1 As expected body mass index percent body fatand waist and hip circumferences were significantly higherin obese group compared to lean group (119875 lt 00001)

Mediators of Inflammation 5

0

1

2

3

4

0 100 200 300

RAN

TES

(ng

mL)

IL-1ra (pgmL)

IL-1ra

r2 = 042P = 0001

(a)

0

1

2

3

4

RAN

TES

(ng

mL)

00 05 10

IP-10 (ngmL)

IP-10

r2 = 031P = 002

(b)

0

1

2

3

4

RAN

TES

(ng

mL)

0 1 2 3

TBARS (120583M)

TBARS

r2 = 029P = 003

(c)

Figure 1 Correlation analysis of circulating RANTES with key inflammatory makers In a mixed population of lean (119899 = 17) and obese(119899 = 40) circulating RANTES correlated negatively with the IL-1ra levels and positively with IP-10 chemokine levels and TBARS activityCorrelations were assessed using Spearmanrsquos rank correlation coefficient

Obese subjects had also higher systolic blood pressure andhigher levels of triglycerides (119875 lt 005) however there wasno significant difference in the cardiopulmonary parametersbetween the two groups Obese subjects had higher levels ofglucose and HbA1c (119875 lt 005) in spite of not being diabeticCompared to lean group the metabolic and inflammatoryprofiles are dysregulated in obese group as indicated byincreased levels of leptin (119875 = 00006) resistin (119875 = 004)IP-10 (119875 = 00008) and RANTES chemokines (119875 = 0023)No significant difference was observed in the other metabolicand inflammatory mediators as well as the oxidative stressmarkers (Table 1 and data not shown)

32 Levels of RANTES Protein andmRNAAre Increased in theAdipose Tissue but Not in PBMCs of Obese Subjects In agree-ment with our recent investigation [38] obese subjects hadhigher levels of RANTES in the circulation that correlatednegatively with IL-1ra (1199032 = 042 119875 = 0001) and positi-vely with IP-10 (1199032 = 031 119875 = 002) and TBARS levels(1199032 = 029 119875 = 003) (Figure 1(a)) To investigate the endo-genous expression of RANTES between the two groups atthe protein level we determined its expression in PBMCsand adipose tissue by western blot flow cytometry andimmunohistochemistry (IHC) As shown in Figure 2(a)there was no difference in the expression of RANTES protein

6 Mediators of Inflammation

RANTES

Actin

15

1

05

0

Fold

chan

ges

Fold

chan

ges

P = 0014

Lean

Lean

(n=7

)

Obese

Obe

se(n

=11

)

RANTES

0

1

2

3

4

Lean

Lean

Obese

Obese

00

05

10

15

20

25

00

05

10

15

20

2530

NC Lean ObeseNC Lean ObeseObe

se(n

=11

) Fold

chan

ges i

n IL

-6pr

otei

n P = 0024

Fold

chan

ges i

n TN

F-120572

prot

ein P = 0037

Lean

(n=7

)

Obe

se(n

=11

)Le

an(n

=7

)

IL-6TNF-120572

(f)

Lean Obese

Lean (n = 3)

(a) (b)

Obese (n = 3)

Lean (n = 3)Obese (n = 3)

MFI

(times1000

)

CD14+

CD14++

CD3+

RANTES APC-A RANTES APC-A

RANTES APC-A RANTES APC-A

CD14

PE-

ACD

3 F

ITC-

A

CD14

PE-

ACD

3 F

ITC-

A

105104103102

105104103102

105104103102

105104103102

105

104

103

102

105

104

103

102

105

104

103

102

105

104

103

102

0

1

1

2

2

3

P = 03

0

02

04

06

08

1

12

14

Lean (n = 10)

Obese(n = 10)

mRN

AFo

ld ch

ange

s in

RAN

TES

(PBM

Cs)

mRN

AFo

ld ch

ange

s in

RAN

TES

(adi

pose

tiss

ue)

P = 002

0

05

1

15

2

25

LeanObese

LeanObese

(e)(c) (d)

Leann = 7)(

Obesen = 11)(

Figure 2 Continued

Mediators of Inflammation 7

P = 001

P = 002

Lean (n = 7)Obese (n = 11)

0

05

1

15

2

25

TNF-120572 IL-6

Fold

chan

ges i

n m

RNA

expr

essio

n(a

dipo

se ti

ssue

)(g)

Figure 2 Expression of RANTES in obese subjects (a)Western blot analysis of RANTES expression in PBMCs from lean and obese subjectsThe bands reacting with anti-RANTES antibody were quantified as described in Section 2 and the relative intensity was determined aftercorrection with Actin that was used as internal control to monitor loading efficiencyThe blots shown are representative of three independentexperiments with consistent results The data are presented in the form of a bar graph on the right of the figure as fold changes of RANTESprotein expression in obese compared to lean subjects (b) Characterization of the monocyte subpopulations and T cells in peripheralblood from lean and obese participants Monocytes subsets were defined by staining for CD14 (PE) T cells by CD3 (FITC) and expressionof intracellular RANTES (APC) was analyzed Left and right upper panels are representative dot plots of CD3 and RANTES expressiononin T cells from lean and obese participants respectively Left and right lowest panels are representative dot plots of CD14 and RANTESexpression onin monocyte subsets from lean and obese participants respectively The double-positive populations (ie CD3+RANTES+CD14+RANTES+ and CD14++RANTES+) were analyzed for mean RANTES fluorescence intensity (c) Analysis of RANTES expression byimmunohistochemistry (IHC) in the subcutaneous adipose tissues from lean and obese nondiabetic participants Aperio software was usedto quantify positive staining (indicated by arrows) and quantified values relative to lean controls are plotted in a bar graph at the bottom (dand e) Analysis of RANTES mRNA expression by quantitative real-time PCR (qRT-PCR) between lean and obese subjects Total RNA wasisolated from PBMCs (d) and adipose tissue biopsies (e) The data are presented as fold changes in obese compared to lean subjects afternormalization with the GAPDH reference gene (f and g) Analysis of TNF-120572 and IL-6 expression at the protein level by IHC (f) and at themRNA level by qRT-PCR (g) in the adipose tissue from lean and obese subjects In IHC experiments Aperio software was used to quantifypositive staining as indicated above and the values are illustrated at the bottom as fold changes compared to lean Mann-Whitney test wasused to determine significance of difference between the lean and obese subjects For each experiment the sample size from each group isindicated by 119899

in PBMCs between lean and obese subjects (119899 = 3 foreach group) Consistent with this finding flow cytometryindicated that RANTES levels were comparable betweenlean and obese subjects (119899 = 3 for each group) in bothmonocytes and lymphocytes (Figure 2(b)) By contrast toPBMCs the expression of RANTES protein in the adiposetissue asmonitored by IHC studies was significantly higher inobese (119899 = 11) compared to lean (119899 = 7) subjects (119875 = 001Figure 2(c)) The differential expression of RANTES betweenPBMCs and adipose tissue in lean and obese subjects wasalso confirmed at the mRNA level by using quantitative real-time PCR (qRT-PCR) and the results are displayed in Figures2(d) and 2(e) Accordingly there was no change in RANTESmRNA levels in PBMCs from lean and obese subjects (119899 =10 for both lean and obese Figure 2(d)) whereas the levelsof RANTES mRNA in the adipose tissue were significantlyhigher in obese (119899 = 11) compared to lean (119899 = 7) subjects(119875 = 002 Figure 2(e)) Under the same conditions theendogenous expression of the two key inflammatory markersTNF-120572 and IL-6 in the adipose tissue were significantly

increased in obese subjects both at the protein level (119875 lt 005Figure 2(f)) and mRNA level (119875 lt 005 Figure 2(g))

33 CCR5mRNA Is Upregulated in the Adipose Tissue of ObeseSubjects We next examined the expression profile of CCR5receptor between lean and obese subjects using PBMCs andadipose tissue Data shown in Figure 3 indicated a peculiarexpression pattern of CCR5 receptor Indeed there was aclear upregulation of Ccr5 mRNA in the adipose tissue ofobese (119899 = 11) compared to lean (119899 = 7) subjects (119875 =003 Figure 3(b)) By contrast Ccr5mRNA was significantlydownregulated in PBMCs of obese subjects compared to leansubjects (119899 = 10 for both lean and obese) (119875 = 004Figure 3(a)) On the other hand flow cytometry analysiscarried out on PBMCs from lean and obese (119899 = 5 for eachgroup) revealed a significant and unique increase of CCR5in CD14+ monocyte subset from obese subjects (119875 = 002Figure 3(c)) The specific mean fluorescence intensity forCCR5 in obese individuals revealed a higher signal for CD14+

8 Mediators of Inflammation

Lean (n = 10)

Obese(n = 10)

14

12

1

08

06

04

02

0

P = 004

Fold

chan

ges i

n CC

R5m

RNA

(PBM

Cs)

(a)

Obese(n = 11)

Lean(n = 7)

2181614121

080604020

P = 003

Fold

chan

ges i

n CC

R5m

RNA

(adi

pose

tiss

ue)

(b)

105

104

103

102

105

104

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102

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102

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CD3

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C-A

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C-A

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PE-

ACD

14

PE-

A

CCR5 APC-ACCR5 APC-A

CCR5 APC-ACCR5 APC-A

Lean Lean

Obese Obese

CD14+ CD14++ CD3+

MFI

(times1000

)

Lean (n = 5)Obese (n = 5)

8

7

6

5

4

3

2

1

0

P = 0001

P = 002

(c)

Figure 3 Differential regulation of CCR5 in PBMCs and adipose tissue is associated with obesity CCR5 gene expression was measuredby qRT-PCR in PBMCs (a) from lean and obese nondiabetic subjects and in the adipose tissue (b) from lean and obese nondiabeticsubjects The data are presented as fold changes in obese compared to lean subjects after normalization with the GAPDH reference gene(c) Characterization of the monocyte subpopulations and T cells in peripheral blood from lean and obese subjects Monocytes subsets weredefined by staining for CD14 (PE) T cells by CD3 (FITC) and expression of CCR5 (APC) and then were analyzed Gates P4 and P3 definethe CD14+ and CD14++ subsets respectively Left and right upper panels are representative dot plots of CD3 and CCR5 expression on Tcells from lean and obese subjects respectively Left and right lower panels are representative dot plots of CD14 and RANTES expressionon monocyte subsets from lean and obese participants respectively The double-positive populations (ie CD3+CCR5+ CD14+CCR5+ andCD14++CCR5+) were analyzed for mean CCR5 fluorescence intensity For each experiment the sample size from each group is indicated by119899

monocytes compared to CD14++ monocytes (119875 = 0001Figure 3(c)) Lean controls displayed the same trend but toa lesser extent

34 Effect of Physical Exercise on RANTES and CCR5 Expres-sion Wepreviously reported the effectiveness of our physicalexercise protocol on improving the physical clinical andmetabolic parameters on obese subjects [38] Accordinglythere was a significant reduction of PBF and SBP and increasein 119881O

2Max along with a decrease in TBARS levels and a

reduction of inflammatory markers TNF-120572 and IL-6 in thecirculation [38] However physical exercise did not reducethe levels of RANTES in the circulation [38] To investigate

whether physical exercise has an impact on the endogenousexpression of RANTES and CCR5 qRT-PCR and IHC werecarried out on adipose tissue from obese subjects before andafter the exercise program As shown in Figure 4(a) IHCcarried out on adipose tissue from obese subjects before(119899 = 11) and after (119899 = 7) the exercise program indicated asignificant decrease in the expression of RANTES by physicalexercise (119875 = 0003) qRT-PCR performed on obese beforeand after the exercise program (119899 = 10 for each group)confirmed the reduction of RANTES mRNA expression byphysical exercise (119875 = 001 Figure 4(b)) Likewise CCR5mRNA was significantly reduced by physical exercise inthe adipose tissue (119875 = 002 Figure 4(b)) Using the

Mediators of Inflammation 9

Obese(before exercise n = 11)

Obese

RANTES

(after exercise n = 7)

P = 0003

Obese (before)

Obese (after)

15

1

05

0

Fold

chan

ges

(a)

Obese (before exercise n = 10)Obese (after exercise n = 10)

RANTES CCR5 TNF-120572 IL-6

P = 001 P = 001

P = 002 P = 004

Fold

chan

ges i

n RA

NTE

S m

RNA

(a

dipo

se ti

ssue

)

25

2

15

1

05

0

(b)

Obese (before exercise n = 11)(after exercise n = 7)Obese

TNF-120572IL-6

Fold

chan

ges i

n pr

otei

n ex

pres

sion

(adi

pose

tiss

ue)

p-JNK

P = 001P = 002 P = 0002

15

1

05

0

(c)

Figure 4 Physical exercise reduces the expression of RANTES andCCR5 in the adipose tissue (a) IHC stainingwith RANTES antibody usingsubcutaneous adipose tissues from obese subjects before and after 3months of physical exercise Aperio software was used to quantify positivestaining in obese before and after physical exercise The data are plotted in a bar graph on the right of the figure as fold changes of RANTESprotein expression in obese subjects before and after the physical exercise program (b) qRT-PCR analysis of RANTES CCR5 TNF-120572 andIL-6 mRNA expression in the adipose tissue from obese before and after 3 months of physical exercise (c) Graphic representation of IHCstaining with TNF-120572 anti-IL-6 and anti-Phospho-JNK antibodies using subcutaneous adipose tissues from obese subjects before and after3 months of physical exercise Aperio software was used to quantify positive staining in obese before and after physical exercise Paired t-testfor two group analysis was done to compare the expression of proteins and mRNA in obese before and after exercise For each experimentthe sample size from each group is indicated by 119899

same samples we observed a decrease in the endogenousexpression of TNF-120572 and IL-6 both at the mRNA level (119875 lt005 Figure 4(b)) and protein level (119875 lt 005 Figure 4(c))

Since obesity is known to induce the phosphorylation ofJNK protein we measured the levels of phosphorylated JNKin adipose tissue by IHC before (119899 = 11) and after (119899 = 7)the physical exercise program Data shown on Figure 4(c)indicated that physical exercise decreases significantly thelevels of phosphorylated JNK (119875 = 0002) in obese in amanner that was concomitantwith a decrease of CCR5 and itsligand RANTES No effect was observed for total JNK beforeand after exercise in obese subjects (data not shown) Takentogether these data suggest that exercise is interfering withobesity-mediated expression of RANTES and CCR5

4 Discussion

Chronic low-grade inflammation state is a key feature ofobesity and it is caused mainly by infiltration of macrophagesand other inflammatory cells into the adipose tissue Theemerging role of RANTES signaling pathway to this chroniccondition is well established The present study explored theeffect of physical exercise on the expression of RANTESand its main receptor Ccr5 in obese humans Our focus onCCR5 receptor was based on a recent study in which CCR5knockout mice were protected from obesity-induced adiposetissue inflammation and insulin resistance [24] In additionit has been previously shown that obesity triggers a modestchange in the expression of CCR3 and no change in that of

10 Mediators of Inflammation

CCR1 [17] The main findings of our current investigationare as follows (1) the expression of both RANTES and CCR5was significantly higher in the subcutaneous adipose tissueof obese subjects (2) by contrast to the adipose tissue Ccr5was downregulated in PBMCs of obese subjects comparedto lean subjects but there was no significant difference inthe expression of RANTES between the two groups and (3)physical exercise corrected the dysregulated expression ofboth RANTES and CCR5 in the subcutaneous adipose tissuebut has a marginal effect on circulating levels of RANTESWhile our findings showing increased expression of RANTESand CCR5 in the adipose tissue are consistent with previousclinical and animal studies [17 40] the downregulation oftheir expression by physical exercise is novel Based on thecrucial role of RANTESCCR5 signaling pathway in thepathology of obesity and its associated complications ourfindings add further evidence that physical exercise mightbe one of nonpharmacologic approaches that can attenuateRANTESCCR5 signaling pathway and thereby mitigatingthe inflammatory and metabolic stress triggered by obesity

RANTES is a powerful proinflammatory chemokine thatcontrols the trafficking of immune inflammatory cells suchas monocytes macrophages Th1 T cells and dendritic cellsfrom the circulation into various tissues including adiposetissue [3 9] Previous studies carried out on obese humansand rodents reported high levels of RANTES as well asincreased frequency ofmacrophages in the adipose tissue thatwere concomitant with increased inflammatory responseinsulin resistance and type 2 diabetes [5 17 41ndash44] Ourfindings showing high levels of RANTES in the adiposetissue of obese subjects at both mRNA and protein levelscorroborate these pioneer studies that associated RANTESwith obesity Unlike the adipose tissue the levels of RANTESwere however comparable between lean and obese subjectsas monitored by western blotting flow cytometry and qRT-PCR The mechanism underlying this differential expressionof RANTES between PBMCs and adipose tissue remainsto be investigated but there is evidence for depot-specificdifferences that dictate the levels of RANTES released byvarious adipose tissues in humans [44]

One of the key characteristics of macrophages that infil-trate the adipose tissue is the heterogeneity of their pheno-type and depending on their polarization status they canbe proinflammatory macrophages (M1-phenotype) secretingvarious inflammatory mediators such as TNF-120572 IL-6 andiNOS [45 46] or anti-inflammatory macrophages (M2-phenotype) that secrete anti-inflammatory cytokines such asIL-10 [41 47] In obesity the differentiation of M2 into M1macrophages is considered as a major event that sustainschronic inflammation [5] Indeed studies on mice indicatedthat obesity induces a shift in macrophage balance towardsthe M1-phenotype macrophages that perpetuate further theinflammatory response and insulin resistance [5 46] Inour study population we did not evaluate the status ofM1- and M2-phenotypes which may represent a limitationto this investigation However in our study the high lev-els of circulating RANTES correlated negatively with theanti-inflammatory IL-1ra and positively with the levels of

proinflammatory IP-10 chemokine andTBARS supporting itsrole in mediating inflammation

In the current study we have also investigated theexpression pattern of CCR5 in obese humans and similarto our findings with RANTES both mRNA and proteinlevels of Ccr5 were increased in the adipose tissue of obesecompared to lean group The observed increase is consistentwith recent studies both on humans and rodents [24 40]The direct role of CCR5 in the regulation of obesity-inducedadipose tissue inflammation and development of insulinresistance was recently demonstrated using CCR5 knockoutmice [24] A dominant shift occurred from proinflammatoryM1 to anti-inflammatoryM2macrophage phenotypes whichcontributed to an improved impaired glucose tolerance andinsulin sensitivity in response to diet-induced obesity in theseanimals [24]

Unexpectedly our data demonstrated that unlike theadipose tissue the expression ofCcr5mRNAwas significantlydownregulated in PBMCs from obese individuals To thebest of our knowledge our data is the first to report thedownregulation of Ccr5 mRNA in the PBMCs of obeseindividuals This observed downregulation is intriguing andmight suggest a complex regulation process of its tran-scription The altered expression of CCR5 in PBMCs hasbeen described in rheumatoid arthritis [48] and in womenwith systemic lupus erythematosus [49] It is possible thatother receptors for RANTES such as CCR1 and CCR3 maybe compensating for the reduced expression of CCR5 inobese subjects Interestingly CCR5 expression on CD14+monocyte subset appears to be upregulation in compari-son with CD14++ subset in obese individuals CD14+ cellsare known to exhibit a macrophage-like phenotype withenhanced antigen-presenting capacities higher endothelialaffinity and they are potent producers of proinflammatorycytokines [50] thus they may contribute to the developmentof chronic inflammation

Of particular interest our study emphasized more onthe role of physical exercise in regulating the expressionof RANTES and its receptor CCR5 It is well establishedthat exercise reduces the risk of chronic metabolic and car-diorespiratory diseases by modulating the inflammatory andstress responses [51 52] Our data showed for the first timethat physical exercise significantly reduced the expressionof both RANTES and Ccr5 receptor in the adipose tissuein a manner that was concomitant with decreased PBF andimproved cardiopulmonary performance as monitored bydecreased SBP and increased119881O

2MaxThe reduced expression

of RANTES and CCR5 by physical exercise was also consis-tent with attenuated inflammatory response as indicated bydecreased TNF-120572 and IL-6 in the circulation and reducedlevels of TBARS Our finding revealed also that exercise-mediated decrease in the expression of RANTES and Ccr5in the adipose tissue was not translated to a decrease in thecirculating levels of RANTES The lack of parallel changesbetween systemic and endogenous levels of RANTES afterexercise raises the question regarding the role of RANTES inthe circulation Earlier study indicated that systemic levels ofRANTES are 100-fold higher than those being released fromany of the adipose tissue depots [44] Therefore it is sensible

Mediators of Inflammation 11

to suggest that while adipose tissue is able to produce thischemokine its contribution to systemic levels may be lesssignificant

Physical exercise is also known to inhibit metabolic stressin part by dephosphorylating and thus inactivating JNKstress kinase [34 53 54] In agreement with these findingsour data indicate that there was a significant increase inthe levels of phosphorylated JNK along with high levels ofRANTES and CCR5 in obese subjects that were all reducedby physical exercise (Figure 4(c)) Whether there is a directcrosstalk between RANTESCCR5 signaling pathway andJNK signaling or not remains to be investigated despite thefact that CCR5 knockout mice displayed attenuated signalingvia NF-120581B and JNK [24]

In conclusion the dysregulation of RANTES and its neg-ative correlation with the metabolic and stress responses aresupporting the pathological role of RANTES in obesity Ourresults also provide strong evidence that physical exercisedownregulates the expression of both RANTES and Ccr5in the adipose tissue These findings give strong supportto the concept that physical exercise can be used as anonpharmacologic approach to mitigate the complicationsassociated with obesity particularly inflammation in part byattenuating RANTESCCR5 signaling in the adipose tissue

Conflict of Interests

The authors declare that they have no conflict of interests

Authorsrsquo Contribution

Engin Baturcam and Jehad Abubaker participated in theconception and design of the study supervision of datacollection data analysis and interpretation of the findingsof the study paper writing and revision Mohammed Dehbiparticipated in the conception and design of the studyand paper writing and revision Ali Tiss Mohamed Abu-Farha andAbdelkrimKhadir performed data analysis FahadAl-Ghimlas conducted the exercise study Irina Al-KhairiPreethi Cherian Naser Elkum Maha Hammad Jeena Johnand Sina Kavalakatt participated in the data collection SamiaWarsame and Cynthia Lehe participated in the data collec-tion KazemBehbehani and SaidDermime helpedwith paperrevision Engin Baturcam and Jehad Abubaker contributedequally to this work

Funding

This work was supported by the Kuwait Foundation for theAdvancement of Sciences (KFAS)

Acknowledgments

The authors would like to thank the staff at the TissueBank and Clinical Laboratory for their assistance throughoutthis study Engin Baturcam and Jehad Abubaker are cofirstauthors

References

[1] H Xu G T Barnes Q Yang et al ldquoChronic inflammation in fatplays a crucial role in the development of obesity-related insulinresistancerdquo Journal of Clinical Investigation vol 112 no 12 pp1821ndash1830 2003

[2] S P Weisberg D McCann M Desai M Rosenbaum RL Leibel and A W Ferrante Jr ldquoObesity is associated withmacrophage accumulation in adipose tissuerdquo Journal of ClinicalInvestigation vol 112 no 12 pp 1796ndash1808 2003

[3] I Harman-Boehm M Bluher H Redel et al ldquoMacrophageinfiltration into omental versus subcutaneous fat across differ-ent populations effect of regional adiposity and the comor-bidities of obesityrdquo Journal of Clinical Endocrinology andMetabolism vol 92 no 6 pp 2240ndash2247 2007

[4] JMOlefsky andC K Glass ldquoMacrophages inflammation andinsulin resistancerdquoAnnual Review of Physiology vol 72 pp 219ndash246 2010

[5] C N Lumeng J L Bodzin and A R Saltiel ldquoObesity induces aphenotypic switch in adipose tissue macrophage polarizationrdquoJournal of Clinical Investigation vol 117 no 1 pp 175ndash184 2007

[6] S E Shoelson J Lee and A B Goldfine ldquoInflammation andinsulin resistancerdquo Journal of Clinical Investigation vol 116 no7 pp 1793ndash1801 2006

[7] S Fujisaka I Usui A Bukhari et al ldquoRegulatory mechanismsfor adipose tissue M1 and M2 macrophages in diet-inducedobese micerdquo Diabetes vol 58 no 11 pp 2574ndash2582 2009

[8] V Appay and S L Rowland-Jones ldquoRANTES a versatile andcontroversial chemokinerdquo Trends in Immunology vol 22 no 2pp 83ndash87 2001

[9] M Keophiphath C Rouault A Divoux K Clement and DLacasa ldquoCCL5 promotes macrophage recruitment and survivalin human adipose tissuerdquo Arteriosclerosis Thrombosis andVascular Biology vol 30 no 1 pp 39ndash45 2010

[10] R Ross ldquoAtherosclerosismdashan inflammatory diseaserdquo The NewEngland Journal of Medicine vol 340 no 2 pp 115ndash126 1999

[11] I Lemieux A Pascot D Prudrsquohomme et al ldquoElevated C-reactive protein another component of the atherothromboticprofile of abdominal obesityrdquo Arteriosclerosis Thrombosis andVascular Biology vol 21 no 6 pp 961ndash967 2001

[12] C-H Tang C-J Hsu and Y-C Fong ldquoThe CCL5CCR5axis promotes interleukin-6 production in human synovialfibroblastsrdquo Arthritis amp Rheumatism vol 62 no 12 pp 3615ndash3624 2010

[13] R F Schwabe R Bataller and D A Brenner ldquoHuman hepaticstellate cells express CCR5 and RANTES to induce proliferationandmigrationrdquoAmerican Journal of Physiology Gastrointestinaland Liver Physiology vol 285 no 5 pp G949ndashG958 2003

[14] E Seki S de Minicis G-Y Gwak et al ldquoCCR1 and CCR5 pro-mote hepatic fibrosis in micerdquo Journal of Clinical Investigationvol 119 no 7 pp 1858ndash1870 2009

[15] J Melchjorsen and S R Paludan ldquoInduction of RANTESCCL5by herpes simplex virus is regulated by nuclear factor 120581B andinterferon regulatory factor 3rdquo Journal of General Virology vol84 no 9 pp 2491ndash2495 2003

[16] S Shahrara A E I Proudfoot J MWoods et al ldquoAmeliorationof rat adjuvant-induced arthritis by Met-RANTESrdquo Arthritis ampRheumatism vol 52 no 6 pp 1907ndash1919 2005

[17] H Wu S Ghosh X D Perrard et al ldquoT-cell accumulation andregulated on activation normal T cell expressed and secretedupregulation in adipose tissue in obesityrdquo Circulation vol 115no 8 pp 1029ndash1038 2007

12 Mediators of Inflammation

[18] P Lusso L Vangelista R Cimbro et al ldquoMolecular engineeringof RANTES peptide mimetics with potent anti-HIV-1 activityrdquoFASEB Journal vol 25 no 4 pp 1230ndash1243 2011

[19] E S Raborn F Marciano-Cabral N E Buckley B R Martinand G A Cabral ldquoThe cannabinoid delta-9-tetrahydrocan-nabinol mediates inhibition of macrophage chemotaxis toRANTESCCL5 linkage to the CB2 receptorrdquo Journal of Neu-roImmune Pharmacology vol 3 no 2 pp 117ndash129 2008

[20] M-L Berres R R Koenen A Rueland et al ldquoAntagonism ofthe chemokine Ccl5 ameliorates experimental liver fibrosis inmicerdquo Journal of Clinical Investigation vol 120 no 11 pp 4129ndash4140 2010

[21] V Braunersreuther C Pellieux G Pelli et al ldquoChemokineCCL5RANTES inhibition reduces myocardial reperfusion inj-ury in atherosclerotic micerdquo Journal of Molecular and CellularCardiology vol 48 no 4 pp 789ndash798 2010

[22] LDumaDHaussingerM Rogowski P Lusso and SGrzesiekldquoRecognition of RANTES by extracellular parts of the CCR5receptorrdquo Journal of Molecular Biology vol 365 no 4 pp 1063ndash1075 2007

[23] M Juremalm N Olsson and G Nilsson ldquoSelective CCL5RANTES-induced mast cell migration through interactionswith chemokine receptors CCR1 and CCR4rdquo Biochemical andBiophysical Research Communications vol 297 no 3 pp 480ndash485 2002

[24] H Kitade K Sawamoto M Nagashimada et al ldquoCCR5 plays acritical role in obesity-induced adipose tissue inflammation andinsulin resistance by regulating both macrophage recruitmentand M1M2 statusrdquo Diabetes vol 61 no 7 pp 1680ndash1690 2012

[25] S N Blair H W Kohl III R S Paffenbarger Jr D G Clark KH Cooper and L W Gibbons ldquoPhysical fitness and all-causemortality a prospective study of healthy men and womenrdquoJournal of the American Medical Association vol 262 no 17 pp2395ndash2401 1989

[26] V B OrsquoLeary C M Marchetti R K Krishnan B P StetzerF Gonzalez and J P Kirwan ldquoExercise-induced reversal ofinsulin resistance in obese elderly is associated with reducedvisceral fatrdquo Journal of Applied Physiology vol 100 no 5 pp1584ndash1589 2006

[27] R Shern-Brewer N Santanam C Wetzstein J White-Welkleyand S Parthasarathy ldquoExercise and cardiovascular disease anew perspectiverdquo Arteriosclerosis Thrombosis and VascularBiology vol 18 no 7 pp 1181ndash1187 1998

[28] M Gleeson N C Bishop D J Stensel M R Lindley S SMastana and M A Nimmo ldquoThe anti-inflammatory effects ofexercise mechanisms and implications for the prevention andtreatment of diseaserdquoNature Reviews Immunology vol 11 no 9pp 607ndash615 2011

[29] N Mathur and B K Pedersen ldquoExercise as a mean to controllow-grade systemic inflammationrdquo Mediators of Inflammationvol 2008 Article ID 109502 6 pages 2008

[30] A M W Petersen and B K Pedersen ldquoThe anti-inflammatoryeffect of exerciserdquo Journal of Applied Physiology vol 98 no 4pp 1154ndash1162 2005

[31] K L Timmerman M G Flynn P M Coen M M Markof-ski and B D Pence ldquoExercise training-induced lowering ofinflammatory (CD14+CD16+) monocytes a role in the anti-inflammatory influence of exerciserdquo Journal of Leukocyte Biol-ogy vol 84 no 5 pp 1271ndash1278 2008

[32] D Vissers W Hens J Taeymans J P Baeyens J Poortmansand L vanGaal ldquoThe effect of exercise on visceral adipose tissue

in overweight adults a systematic review and meta-analysisrdquoPLoS ONE vol 8 no 2 Article ID e56415 2013

[33] N Kawanishi H Yano Y Yokogawa and K Suzuki ldquoExer-cise training inhibits inflammation in adipose tissue via bothsuppression of macrophage infiltration and acceleration ofphenotypic switching fromM1 to M2macrophages in high-fat-diet-induced obese micerdquo Exercise Immunology Review vol 16pp 105ndash118 2010

[34] M A Kiraly J Campbell E Park et al ldquoExercise maintainseuglycemia in association with decreased activation of c-JunNH2-terminal kinase and serine phosphorylation of IRS-1in the liver of ZDF ratsrdquo American Journal of PhysiologyEndocrinology and Metabolism vol 298 no 3 pp E671ndashE6822010

[35] G Caimi B Canino G Amodeo M Montana and R LPresti ldquoLipid peroxidation and total antioxidant status inunprofessional athletes before and after a cardiopulmonarytestrdquo Clinical Hemorheology and Microcirculation vol 43 no3 pp 235ndash241 2009

[36] N P E Kadoglou F Iliadis N Angelopoulou et al ldquoTheanti-inflammatory effects of exercise training in patients withtype 2 diabetes mellitusrdquo European Journal of CardiovascularPrevention and Rehabilitation vol 14 no 6 pp 837ndash843 2007

[37] Z Radak M Sasvari C Nyakas et al ldquoRegular trainingmodulates the accumulation of reactive carbonyl derivatives inmitochondrial and cytosolic fractions of rat skeletal musclerdquoArchives of Biochemistry and Biophysics vol 383 no 1 pp 114ndash118 2000

[38] J Abubaker A Tiss M Abu-Farha et al ldquoDNAJB3HSP-40cochaperone is downregulated in obese humans and is restoredby physical exerciserdquo PLoS ONE vol 8 no 7 Article ID e692172013

[39] K J Livak and T D Schmittgen ldquoAnalysis of relative geneexpression data using real-time quantitative PCR and the 2-ΔΔCT methodrdquoMethods vol 25 no 4 pp 402ndash408 2001

[40] J Huber F W Kiefer M Zeyda et al ldquoCC chemokine andCC chemokine receptor profiles in visceral and subcutaneousadipose tissue are altered in human obesityrdquo Journal of ClinicalEndocrinology and Metabolism vol 93 no 8 pp 3215ndash32212008

[41] S Gordon ldquoAlternative activation of macrophagesrdquo NatureReviews Immunology vol 3 no 1 pp 23ndash35 2003

[42] H Kanda S Tateya Y Tamori et al ldquoMCP-1 contributes tomacrophage infiltration into adipose tissue insulin resistanceand hepatic steatosis in obesityrdquo Journal of Clinical Investigationvol 116 no 6 pp 1494ndash1505 2006

[43] A C Konner and J C Bruning ldquoToll-like receptors link-ing inflammation to metabolismrdquo Trends in Endocrinology ampMetabolism vol 22 no 1 pp 16ndash23 2011

[44] R Madani K Karastergiou N C Ogston et al ldquoRANTESrelease by human adipose tissue in vivo and evidence fordepot-specific differencesrdquo American Journal of PhysiologyEndocrinology andMetabolism vol 296 no 6 pp E1262ndashE12682009

[45] S Gordon and P R Taylor ldquoMonocyte and macrophageheterogeneityrdquo Nature Reviews Immunology vol 5 no 12 pp953ndash964 2005

[46] AMantovani A Sica S Sozzani P Allavena A Vecchi andMLocati ldquoThe chemokine system in diverse forms of macrophageactivation and polarizationrdquo Trends in Immunology vol 25 no12 pp 677ndash686 2004

Mediators of Inflammation 13

[47] J I Odegaard R R Ricardo-Gonzalez A Red Eagle et alldquoAlternativeM2 activation of kupffer cells by PPAR120575 amelioratesobesity-induced insulin resistancerdquo Cell Metabolism vol 7 no6 pp 496ndash507 2008

[48] K J Katschke Jr J B Rottman J H Ruth et al ldquoDifferen-tial expression of chemokine receptors on peripheral bloodsynovial fluid and synovial tissue monocytesmacrophages inrheumatoid arthritisrdquo Arthritis amp Rheumatology vol 44 no 5pp 1022ndash1032 2001

[49] S Bjorkander G Heidari-Hamedani K Bremme I Gunnars-son and U Holmlund ldquoPeripheral monocyte expression ofthe chemokine receptors CCR2 CCR5 and CXCR3 is alteredat parturition in healthy women and in women with systemiclupus erythematosusrdquo Scandinavian Journal of Immunology vol77 no 3 pp 200ndash212 2013

[50] L Ziegler-Heitbrock ldquoThe CD14+ CD16+ blood monocytestheir role in infection and inflammationrdquo Journal of LeukocyteBiology vol 81 no 3 pp 584ndash592 2007

[51] M Takahashi M Miyashita N Kawanishi et al ldquoLow-volumeexercise training attenuates oxidative stress and neutrophilsactivation in older adultsrdquo European Journal of Applied Physi-ology vol 113 no 5 pp 1117ndash1126 2013

[52] N Kawanishi H Yano TMizokamiM Takahashi E Oyanagiand K Suzuki ldquoExercise training attenuates hepatic inflamma-tion fibrosis and macrophage infiltration during diet induced-obesity in micerdquo Brain Behavior and Immunity vol 26 no 6pp 931ndash941 2012

[53] G da Luz M J S Frederico S da Silva et al ldquoEnduranceexercise training ameliorates insulin resistance and reticulumstress in adipose and hepatic tissue in obese ratsrdquo EuropeanJournal of Applied Physiology vol 111 no 9 pp 2015ndash2023 2011

[54] A GOliveira BM Carvalho N Tobar et al ldquoPhysical exercisereduces circulating lipopolysaccharide andTLR4 activation andimproves insulin signaling in tissues of DIO ratsrdquoDiabetes vol60 no 3 pp 784ndash796 2011

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