The effects of voluntary wheel running on neuroinflammatory status: Role of monocyte chemoattractant protein-1

    The effects of voluntary wheel running on neuroinflammatory status: Role of monocyte chemoattractant protein-1 Lindsay J. Spielman, Mehrbod Estaki, Sanjoy Ghosh, Deanna L. Gibson, Andis Klegeris PII: DOI: Reference:

S1044-7431(16)30174-9 doi:10.1016/j.mcn.2016.12.009 YMCNE 3151

To appear in:

Molecular and Cellular Neuroscience

Received date: Accepted date:

29 September 2016 30 December 2016

Please cite this article as: Spielman, Lindsay J., Estaki, Mehrbod, Ghosh, Sanjoy, Gibson, Deanna L., Klegeris, Andis, The effects of voluntary wheel running on neuroinflammatory status: Role of monocyte chemoattractant protein-1, Molecular and Cellular Neuroscience (2017), doi:10.1016/j.mcn.2016.12.009

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ACCEPTED MANUSCRIPT The effects of voluntary wheel running on neuroinflammatory status: Role of monocyte chemoattractant protein-1

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Lindsay J. Spielman a, Mehrbod Estaki a, Sanjoy Ghosh a, Deanna L. Gibson a, Andis

Department of Biology, University of British Columbia Okanagan Campus, 3187 University

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Way, Kelowna, British Columbia, Canada, V1V 1V7

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a

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Klegeris a,*

* Corresponding author at: Department of Biology, University of British Columbia Okanagan

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Campus, 3187 University Way, Kelowna, British Columbia, Canada, V1V 1V7

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E-mail addresses: [email protected] (L. Spielman), [email protected] (M. Estaki),

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Klegeris).

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[email protected] (S. Ghosh), [email protected] (D. Gibson), [email protected] (A.

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ACCEPTED MANUSCRIPT ABSTRACT The health benefits of exercise and physical activity (PA) have been well researched and it is widely accepted that PA is crucial for maintaining health. One of the mechanisms by

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which exercise and PA exert their beneficial effects is through peripheral immune system adaptations. To date, very few studies have looked at the regulation of neuroimmune reactions in

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response to PA. We studied the effect of voluntary wheel running (VWR) on pro- and anti-

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inflammatory cytokine levels, patterns of glial cell activation and expression of immune receptors

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in the brains of female C57BL/6 mice. By using homozygous monocyte chemoattractant protein (MCP)-1 null mice, we investigated the role of this key immunoregulatory cytokine in mediating

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VWR-induced neuroinflammatory responses. We demonstrated that, compared to their sedentary counterparts, C57BL/6 mice exposed for seven weeks to VWR had increased levels of pro- and

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anti-inflammatory cytokines, markers of glial cell activation and a trend towards increased

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expression of toll-like receptor (TLR) 4 in the brain. Measurements of serum cytokines revealed

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that the alterations in brain cytokine levels could not be explained by the effects of PA on peripheral cytokine levels. We propose that the modified neuroimmune status observed in the VWR group represents an activated immune system, as opposed to a less activated immune system in the sedentary group. Since MCP-1 knockout mice displayed differing patterns of proand anti-inflammatory brain cytokine expression and glial activation when compared to their wildtype counterparts, we concluded that the effects of VWR on neuroimmune reactions may be modulated by MCP-1. These identified immunomodulatory effects of PA in the brain could contribute to the observed positive relationship between physically active lifestyles and a reduced risk for a number of neurodegenerative diseases that possess a significant neuroinflammatory component.

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Keywords:

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Brain, Cytokines, Exercise, Glia, MCP-1 knockout, Neuroinflammation,

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ACCEPTED MANUSCRIPT Abbreviations analysis of variance

BBB

blood brain barrier

BDNF

brain-derived neurotropic factor

BSA

bovine serum albumin

CBS

calf bovine serum

CNS

central nervous system

DMEM-F12

Dulbecco’s modified Eagle medium: nutrient mixture F-12 Ham

DMSO

dimethyl sulfoxide

ECL

enhanced chemiluminescent

ELISA

enzyme-linked immunosorbent assay

GDNF

glial cell line-derived neurotrophic factor

GFAP

glial fibrillary acidic protein

H2O2

hydrogen peroxide

HRP

horseradish peroxidase

HSP

heat shock protein

IBA-1

ionized calcium binding adaptor molecule 1

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ANOVA

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ACCEPTED MANUSCRIPT interferon

IL

interleukin

LPS

lipopolysaccharide

LSD

lest significant difference

M1

pro-inflammatory macrophage

M2

anti-inflammatory macrophage

MCP

monocyte chemoattractant protein

MIP

macrophage inflammatory protein

NSERC

Natural Sciences and Engineering Research Council of Canada

PA

physical activity

RIPA

radioimmunoprecipitation assay

SDS

sodium dodecyl sulphate

SED

sedentary

SEM

standard error of the mean

STAT

signal transducer and activator of transcription

TBST

tris-buffered saline with 0.1% tween

TEMED

tetramethylethylenediamine

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IFN

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ACCEPTED MANUSCRIPT toll-like receptor

TNF

tumor necrosis factor

UBC

University of British Columbia

VEGF

vascular endothelial growth factor

VWR

voluntary wheel running

ZO

zonulae occludens

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TLR

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1. Introduction It is generally understood that physical activity (PA) is important for maintaining a healthy

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body. The benefits of both PA (any bodily movement involving the contraction of skeletal muscle

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that increases energy expenditure above the basal level) and exercise (a subcategory of PA that

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involves deliberate, structured and repetitive activity aimed towards enhanced muscular tone or endurance abilities) are well known (CDC, 2015). PA and exercise enhance cardiovascular

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endurance, increase muscular strength, boost metabolism, improve signaling of various hormones and decrease adiposity (Bergman, 2013; Egan and Zierath, 2013; Fiuza-Luces et al., 2013; Peixoto

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et al., 2015; Stewart et al., 2005; Stojanovic et al., 2012). Research has highlighted the overall

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anti-inflammatory effect that exercise and PA produces on the body when compared to a sedentary

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(SED) lifestyle (Gleeson et al., 2011).

Some of the key immune-modulating effects that regular exercise and PA have in the periphery include a reduction in circulating pro-inflammatory cytokines, like tumor necrosis factor (TNF)-α and monocyte chemoattractant protein (MCP)-1 (Bruun et al., 2006; Gleeson et al., 2011; McFarlin et al., 2006; Michigan et al., 2011; Stewart et al., 2005), with an increase in antiinflammatory cytokines including interleukin (IL)-10 (Gleeson et al., 2011; McFarlin et al., 2006; Starkie et al., 2003; Steensberg et al., 2003). These cytokines and chemokines are capable of crossing the blood brain barrier (BBB), and can serve as a means of communication between the peripheral and central nervous system (CNS) immune system (Banks and Erickson, 2010; Banks et al., 1995). There are a number of additional ways in which the CNS and peripheral immune system communicate, including the recently discovered (and still somewhat debated) CNS

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ACCEPTED MANUSCRIPT lymphatic system, extravasation of immune cells across the BBB, and secretion of cytokines and chemokines by brain endothelial cells (Aspelund et al., 2015; Coisne et al., 2013; Louveau et al.,

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2015a; Louveau et al., 2015b; Shoemaker et al., 2014; Verma et al., 2006). PA and exercise may

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alter some or all of the above mechanisms involved in the peripheral to CNS immune system

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communication.

Exercise can also transform adipose tissue resident macrophages from a pro-inflammatory

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(M1) to an anti-inflammatory (M2) state (Bruun et al., 2006). The mechanism behind these

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phenomena is not fully understood, but is thought to involve the downregulated expression of tolllike receptor (TLR) 4 on several cell types, including monocytes, adipocytes, myocytes and

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hepatocytes (Gleeson et al., 2011; McFarlin et al., 2006), leading to a significant reduction of

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inflammation. Overall, while much is known about the effects of PA and exercise on peripheral

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immunity, the effects of PA on the brain immune system are relatively unknown. PA and exercise can have several beneficial effects on functions of the CNS, such as improved mood and mental health, as well as enhanced memory and cognitive function (Moore et al., 2014; Roig et al., 2013). Exercise is known to enhance neuronal release of the neurotransmitters serotonin and dopamine (Melancon et al., 2014; Monteiro-Junior et al., 2015). Exercise has also been associated with enhanced long-term potentiation and neurogenesis (Yu et al., 2013), as well as higher expression of CNS cell survival signals, like brain-derived neurotropic factor (BDNF), vascular endothelial growth factor (VEGF) and glial cell line-derived neurotrophic factor (GDNF) (Gyorkos et al., 2014; Uysal et al., 2015). However, only limited information is available regarding the effects of exercise and PA on glia, the immune and helper cells of the brain.

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ACCEPTED MANUSCRIPT In this study, we used voluntary wheel running (VWR) mice to examine the effects of PA on brain cytokine production and glial cell activation. We also studied the role that MCP-1 plays

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in these responses, since previous research has shown that MCP-1 mediates cross talk between the peripheral immune system and the immune system of the brain. MCP-1 is an extracellularly

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secreted chemokine, which is capable of crossing the BBB (Erickson and Banks, 2011). It is

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produced by many peripheral and CNS cell types, including smooth muscle cells, fibroblasts,

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monocytes, astrocytes and microglia (Deshmane et al., 2009).

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This immune cytokine is upregulated during pathological events in both the periphery and the CNS, including arthritis, cardiovascular disease, multiple sclerosis and Alzheimer’s disease

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(Deshmane et al., 2009). More specifically, MCP-1 has been shown to increase permeability of the

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BBB (Paul et al., 2014) by downregulating the expression of the tight junction proteins zonulae occludens (ZO)-1 and occludin by brain endothelial cells (Song and Pachter, 2004). This leads to

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increased extravasation of immune cells across the BBB (Carrillo-de Sauvage et al., 2012; Sewell et al., 2004) and facilitates increased crosstalk between the peripheral and CNS immune systems. Our results demonstrate that VWR upregulates expression of several pro- and antiinflammatory cytokines in the brain, activates glial cells and induces changes in CNS TLR4 expression. We also demonstrate that the neuroimmune-modulatory effects of VWR in the CNS are at least partially mediated by MCP-1. Furthermore, we demonstrate that the changes in brain cytokine levels in response to VWR appear to be independent of changes in circulating peripheral cytokine levels, indicating that PA may have unique and brain-specific immune-modulatory roles.

2. Methods

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ACCEPTED MANUSCRIPT 2.1. Materials Ammonium persulfate, dimethyl sulfoxide (DMSO), ExtrAvidin alkaline phosphatase,

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phosphatase substrate tablets, protease inhibitor cocktail, sodium deoxycholate,

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tetramethylethylenediamine (TEMED) and Triton X-100 were obtained from Sigma Aldrich

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(Oakville, ON, Canada). Bromophenol blue was obtained from Van Waters and Rogers International (Mississauga, ON, Canada). Murine enzyme-linked immunosorbent assay (ELISA)

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development kits for MCP-1, IL-4, IL-10, TNF-α and interferon (IFN)-γ were purchased from

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PeproTech (Rocky Hill, NJ, USA). Acrylamide/bis (29:1, 30% solution), bovine serum albumin (BSA), calf bovine serum (CBS), Dulbecco’s modified Eagle medium: nutrient mixture F-12 Ham

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(DMEM-F12), diethanolamine, glycine, hydrochloric acid, penicillin/streptomycin, Pierce BCA

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protein assay kit, sodium dodecyl sulphate (SDS) and SuperSignal West Pico enhanced chemiluminescent (ECL) substrate were purchased from ThermoFisher Scientific (Ottawa, ON,

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Canada). Precision Plus Protein Kaleidoscope ladder was purchased from Bio-Rad (Mississauga, ON, Canada). DAKO rabbit anti-glial fibrillary acidic protein (GFAP) antibodies were purchased through Cedarlane (Burlington, ON, Canada). Rabbit anti-ionized calcium binding adaptor molecule 1 (IBA-1) antibodies were purchased from WAKO (Irving, CA, USA). Rabbit anti-actin antibody, anti-TLR4 and anti-signal transducer and activator of transcription (STAT) 1 antibodies were purchased from SantaCruz Biotechnology (San Jose, CA, USA). Horseradish peroxidase (HRP) labelled goat anti-rabbit antibody was purchased from Cell Signaling (Orange County, CA, USA).

2.2. Mice

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ACCEPTED MANUSCRIPT Female C57BL/6 mice (termed MCP-1+/+) and female MCP-1 knockout (MCP-1-/-) mice on a C57BL/6 background were purchased from Jackson Laboratory (Bar Harbor, Maine, USA).

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Mating pairs were set up to breed in house at the Modified Barrier Facility at the University of British Columbia (UBC). All procedures involving the care and handling of the mice were

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approved by the UBC Committee on Animal Care, under the guidelines of the Canadian Council

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on the Use of Laboratory Animals. Mice were housed individually in “shoebox” style cages

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(Columbus Instruments) in a temperature-controlled room (22°C) on a 12 h light/dark cycle with access to food and water ad libitum. At the age of 6 weeks, mice were randomly divided into two

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groups: 1) VWR (with access to free running wheel (wheel diameter of 10.16 cm, interior diameter of 9.2 cm, wheel width of 5.1 cm, Columbus Instruments), as a model for PA and 2)

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SED (with no access to free running wheel). All mice were placed in their respective cages for 3

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days prior to ‘day 1’ of the experiment to acclimate to the wheels and solitude. Mice were housed

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in these conditions for seven weeks. There were four experimental groups in total: MCP-1+/+ SED (N = 8), MCP-1+/+ VWR (N = 8), MCP-1-/- SED (N = 3) and MCP-1-/- VWR (N = 7). Only three animals were available for the MCP-1-/- SED group. It is important to note that previous research determined that housing mice individually can have an impact on certain aspects of neuroinflammation (Karelina et al., 2009); however, it was necessary to house mice in solitude to preclude competition for access to the free running wheel, and to prevent fighting associated with social ranking, which can produce a considerable stress response (Kinsey et al., 2008). To check that the mice were, in fact, performing PA, sample wheel rotation numbers were collected at 1 h intervals for the first two weeks of the seven-week experimental period. Wheel rotations were counted by magnetic switches interfaced to a computer using windows software. SED mice were intentionally housed in absence of a locked free running

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ACCEPTED MANUSCRIPT wheel, since studies have demonstrated that rodents climb and play on locked wheels (Koteja et al., 1999), providing a form of PA. Following the seven-week period, mice were anesthetized with

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isoflurane, blood was collected via cardiac puncture, and mice were sacrificed by cervical

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2.3. Brain tissue dissection and protein extraction

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dislocation. Mice brain tissue and serum were collected and stored at -80°C.

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Brain tissues were dissected as previously described (Chiu et al., 2007). The frontal cortex, cerebellum and hippocampal areas were separated under the dissecting microscope. Brain sections

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were placed into microtubes containing 500 μl radioimmunoprecipitation assay (RIPA) buffer

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with 5 μl of protease inhibitor cocktail. The brain sections were homogenized, incubated on ice for 15 min and then centrifuged at 32,800 x g at 4°C for 15 min. Protein concentrations in all samples

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were measured by the Pierce BCA protein kit as per the manufacturer’s instructions. Working stock of protein (1 mg/ml protein) in RIPA buffer was prepared and stored at -20°C.

2.4. Enzyme linked immunosorbent assay (ELISA) ELISAs for MCP-1, TNF-α, IL-10, IL-4 and IFN-γ were performed as per the manufacturer’s instructions (PeproTech), and the detection limits were experimentally determined to be 0.9, 0.1, 0.6, 0.1 and 0.5 pg/g of wet tissue, respectively.

2.5. Western blots

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ACCEPTED MANUSCRIPT 100 μg of frontal cortex mice brain protein was denatured in 1x loading buffer at 70°C for 5 min and ran on a 12% SDS polyacrylamide gel. Protein samples were transferred for 5 h at 60 V

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onto a nitrocellulose membrane, which was blocked with 5% BSA in tris-buffered saline with 0.1% tween (TBST), and incubated with primary antibodies at optimal dilutions (GFAP 1:20,000;

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IBA-1 1:2,000; TLR4 1:500; STAT1 1:1,000; or actin 1:500) in 3% BSA/TBST overnight.

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Membranes were washed with TBST for 10 min 3x and incubated with secondary anti-rabbit

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antibody (1:10,000) in 3% skim milk powder/TBST for 2 h. Membranes were washed again as before, incubated in ECL reagent for 5 min, imaged using Fluorochem Q image system and

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analyzed with ImageJ software (National Institute of Health, USA). A common protein sample “MCP-1+/+ SED 1” was run on each gel to serve as a visual indicator of proper sample loading,

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protein transfer and antibody binding efficiency. Densitometry values obtained from this common

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control sample band were used to normalize band density values between different blots (Taylor

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and Posch, 2014). Subsequently, intensities of individual bands were normalized against the level of actin, the housekeeping gene, which was measured in the same samples after stripping of the membranes. Western blotting guidelines described by Taylor and Posch (2014) were followed to minimize errors associated with variation in sample loading and protein transfer.

2.6. Serum cytokine measurements Cytokine concentrations in mouse serum samples were measured by Eve Technologies (Calgary, Canada) using multiplex assays detection limits of which were reported to be 0.1 pg/ml.

2.7. BV-2 cell culture experiments 13

ACCEPTED MANUSCRIPT The murine BV-2 microglia cell line was obtained from Dr. G. Garden (Center on Human Development and Disability, University of Washington, Seattle, WA, USA). Cells were cultured

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in DMEM-F12 supplemented with 10% CBS, penicillin (100 U/ml) and streptomycin (100 μg/ml) in T-75 flasks incubated at 37°C in humidified 5% CO2 and 95% air atmosphere. BV-2 cells were

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added to 24-well plates at a concentration of 5 x 105 cells/ml in 0.9 ml of DMEM-F12 containing

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5% CBS. To model the effects observed in vivo, anti-murine MCP-1 antibody was added to the

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BV-2 cells in culture 30 min prior to the addition of 100 μM hydrogen peroxide (H2O2), which was used to model oxidative stress associated with PA (Vogt and Hoppeler, 2010). Following 48 h

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2.8. Statistical analysis

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incubation, supernatants were collected for ELISA.

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Data obtained were analyzed using Shapiro-Wilk normality test, Student’s unpaired t-test and one-way analysis of variance (ANOVA), followed by Tukey’s or Dunnett’s post-hoc test. Average wheel running measurements were log transformed, since daily running averages were not normally distributed for all groups (MCP-1+/+ VWR group Shapiro-Wilk normality test, P = 0.02). Data are presented as means ± standard error of the mean (SEM). Significance was established at P < 0.05. Statistical analyses were not performed on the data obtained from the MCP-1 -/- SED group, due to the low number of animals (N = 3) in this group. The preliminary data acquired from the MCP-1 -/- SED group are presented for reference.

3. Results

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ACCEPTED MANUSCRIPT 3.1. The effects of MCP-1 on VWR behavior in mice There was no significant difference in VWR activity between MCP-1+/+ VWR mice and

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MCP-1-/- VWR mice over the first two weeks of the study (P = 0.23). Thus, this evidence indicates

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the MCP-1 does not affect VWR behavior in mice.

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Fig. 1. Average wheel revolutions per day in VWR groups of MCP-1+/+ and MCP-1-/- mice. Data points represent the log transformed daily average of number of wheel revolutions per individual mouse in weeks one and two (horizontal bars represent means ± SEM). Differences between the VWR groups during weeks one and two were not statistically significant, according to Student’s unpaired t-test (P = 0.23).

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3.2. The effects of VWR on brain cytokine levels are mediated by MCP-1 Changes in the levels of five different cytokines in three different areas of the brain were

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measured (Fig. 2) and are summarized in Figure 3. Concentrations of MCP-1, TNF-α, IL-10 and

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IL-4 were elevated in most of the brain regions of the MCP-1+/+ VWR mice, when compared to MCP-1+/+ SED mice. Similarly, the MCP-1-/- VWR mice displayed elevated brain concentrations

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of TNF-α and IL-4 compared to the MCP-1+/+ SED group, but IL-10 could not be detected in the

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brains of MCP-1-/- VWR. IFN-γ expression in the brains of MCP-1-/- VWR mice was upregulated in comparison to both MCP-1+/+ SED and MCP-1+/+ VWR mice, which was not the case for the

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MCP-1+/+ VWR mice (Fig. 2). The data obtained from the MCP-1 -/- SED animals (N=3) are

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included in the Fig. 2, even though statistical analysis was not performed using these data, to

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illustrate that baseline brain cytokine levels of the animals from the MCP-1 -/- SED group were similar to those from the MCP-1 -/- VWR group. Exceptions included TNF-α, which appeared to be at higher levels in the brains of MCP-1 -/- SED animals compared to their wild-type counterparts, while IFN-γ was not detected in the brains of MCP-1-/- SED mice, but was present at low concentrations in MCP-1+/+ SED mice.

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Fig. 2. Concentrations of MCP-1 (A), TNF-α (B), IL-10 (C), IL-4 (D) and IFN-γ (E) in the frontal cortex, cerebellum and hippocampus of MCP-1+/+ and MCP-1-/- mice following exposure to seven weeks of SED or VWR conditions. Data (means ± SEM) from 3-8 mice per experimental group are presented. The detection limits of the ELISAs are shown as dotted lines; n.d. = not detected. a

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ACCEPTED MANUSCRIPT P < 0.05 compared to the respective brain section from MCP-1+/+ SED mice; b P < 0.05 compared to the respective brain section from MCP-1+/+ VWR mice, according to the one-way ANOVA

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followed by Tukey’s post-hoc test. P and F values for the main effects of the one-way ANOVA are also shown. One-way ANOVA did not include data from the MCP-1-/- SED group, due to the

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low number of mice in this group (N=3). Post-hoc analyses were not performed with values below

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the detection limit.

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Fig. 3. Overview of the changes in the brain cytokine levels of MCP-1+/+ and MCP-1-/- mice in response to VWR.

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3.3. The effects of VWR on astrocytes and microglia: The role of MCP-1 Once we discovered that VWR increased the expression of both pro- and anti-

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inflammatory cytokines in the brains of MCP-1+/+ mice, and that MCP-1-/- VWR mice had a

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differing pattern of brain cytokine expression compared to MCP-1+/+ VWR mice, we sought to

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investigate the activation status of the brain glial cells, which could be the source of these brain cytokines. Western blot analysis confirmed that VWR enhanced expression of GFAP, an

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astrocytic cell activation marker in the frontal cortex of MCP-1+/+ mice (Fig. 4). However, VWR did not cause activation of microglia, as evident by the lack of statistically significant upregulation

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in brain IBA-1 expression in MCP-1+/+ VWR mice compared to MCP-1+/+ SED mice (P = 0.41,

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compared to MCP-1+/+ mice.

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Tukey’s post-hoc test) (Fig. 4). The MCP-1-/- mice had lower levels of both GFAP and IBA-1,

Fig. 4. Representative bands of GFAP, IBA-1 and actin expression in the frontal cortex of SED and VWR groups of MCP-1+/+ and MCP-1-/- mice (A). Densitometric analysis of GFAP (B) and IBA-1 (C) protein expression levels (means ± SEM) from 3-5 mice normalized against those of actin. a P < 0.05 compared to MCP-1+/+ SED mice; b P < 0.05 compared to MCP-1+/+ VWR mice, according to the one-way ANOVA followed by Tukey’s post-hoc test. P and F values for the main

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ACCEPTED MANUSCRIPT effects of the one-way ANOVA are also shown. One-way ANOVA did not include data from the

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MCP-1-/- SED group, due to the low number of mice in this group (N=3).

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3.4. The effects of VWR on TLR4 expression in the brains of mice: The role of MCP-1 Since the anti-inflammatory effects of PA in the periphery could involve systemic

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decreases in the expression of TLR4 (Gleeson et al., 2011; McFarlin et al., 2006), we investigated

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whether the observed immune-modifying effects of VWR in the brain were associated with

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changes in the TLR4 levels. Western blot analysis revealed that, contrary to what has been previously observed in the periphery (Gleeson et al., 2011; McFarlin et al., 2006), only a trend

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towards increased TLR4 expression was observed in MCP-1+/+ VWR mice compared to their SED counterparts (P = 0.07, Tukey’s post-hoc test) (Fig. 5). No difference in TLR4 expression was

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(Fig. 5).

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observed in MCP-1-/- VWR mice, compared to either MCP-1+/+ SED or MCP-1+/+ VWR groups

Fig. 5. Representative bands of TLR4 and actin protein expression in the frontal cortex of SED and VWR groups of MCP-1+/+ and MCP-1-/- mice (A). Densitometric analysis of TLR4 protein expression levels (means ± SEM) from 3-4 mice normalized against those of actin (B). No significant differences were detected according to the one-way ANOVA followed by Fisher’s LSD post-hoc test. P and F values for the main effects of the one-way ANOVA are also shown.

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ACCEPTED MANUSCRIPT One-way ANOVA did not include data from the MCP-1-/- SED group, due to the low number of

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mice in this group (N=3).

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3.5. The effects of VWR on STAT1 expression in the brains of mice: The role of MCP-1 Since STAT1 is significantly and transiently upregulated in myocytes following exercise

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(Begue et al., 2013), and has been associated with infiltration of immune cells across the BBB

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(Chaudhuri et al., 2008), we sought to investigate whether STAT1 expression was altered in the brain following VWR. We found that total STAT1 expression in the frontal cortex of MCP-1+/+

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mice was not affected by VWR (Fig. 6). However, MCP-1-/- VWR mice had significantly lower

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levels of total STAT1, compared to MCP-1+/+ VWR mice (Fig. 6).

Fig. 6. Representative bands of STAT1 and actin expression in the frontal cortex of SED and VWR groups of MCP-1+/+ and MCP-1-/- mice (A). Densitometric analysis of STAT1 protein expression levels (means ± SEM) from 3-4 mice normalized against those of actin (B). b P < 0.05 compared to MCP-1+/+ VWR mice, according to the one-way ANOVA followed by Tukey’s posthoc test. P and F values for the main effects of one-way ANOVA are also shown. One-way ANOVA did not include data from the MCP-1-/- SED group, due to the low number of mice in this group (N=3).

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3.6. Levels of serum cytokines in VWR and SED mice Since the observed changes in brain cytokine levels could be due to altered levels of

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peripheral blood cytokines caused by VWR, we analyzed mice serum levels of the pro- and anti-

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inflammatory cytokines studied. No significant differences in serum concentrations of TNF-α, IL-

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10, IL-4 or IFN-γ, except for between MCP-1+/+ SED and MCP-1-/- VWR for IL-4 (P = 0.02), were detected between any of the mice groups (Table 1). Thus, our data indicate that neither VWR, nor

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the presence or absence of MCP-1 had a detectable effect on the peripheral levels of these

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Table 1

Serum cytokine levels in SED and VWR groups of MCP-1+/+ and MCP-1-/- mice a, b, c. TNF-α

IL-10

IL-4

IFN-γ

(pg/ml)

(pg/ml)

(pg/ml)

(pg/ml)

MCP-1+/+ SED

1.75 ± 0.96

1.48 ± 0.43

0.55 ± 0.27*

n.d.

MCP-1+/+ VWR

1.81 ± 0.74

1.23 ± 0.53

0.42 ± 0.04

n.d.

MCP-1-/- SED

1.05 ± 0.32

0.32 ± 0.33

0.27 ± 0.1

n.d.

MCP-1-/- VWR

1.37 ± 0.33

0.77 ± 0.24

0.21 ± 0.02

n.d.

a

Data are presented as means ± SEM from 3-4 mice

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n.d. = not detected

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Main effects of the one-way ANOVA for TNF-α: P = 0.92, F = 0.08; for IL-10: P = 0. 76, F =

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0.49; for IL-4: P = 0.05, F = 4.25. One-way ANOVA did not include data from the MCP-1-/- SED

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group, due to the low number of mice in this group (N=3).

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*significantly different from MCP-1-/- VWR

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3.7. Secretion of cytokines by cultured BV-2 mouse microglia in response to reduced MCP-1

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levels

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Cell culture studies were used to confirm the role of MCP-1 as a modulator of glial cell

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cytokine secretion. H2O2 was chosen as the microglial stimulus, since it mimics the oxidative

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stress and production of reactive oxygen species that follows PA (Radak et al., 2007). The effect of PA on the brain redox balance is far more complex than simply applying H2O2 to cultured brain

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cells; nevertheless, we chose this model since H2O2 has been shown to be produced by mitochondria in rodent brains (60-350 pmol/min/mg of protein) (Patole et al., 1986) and its

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concentration increases following exercise (Afzalpour et al., 2015). H2O2 was applied to cultured

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cells on its own in the absence of inflammatory stimuli, additional oxidative stressors or anti-

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oxidant compounds that are known to be enhanced in the brain following PA. This was done in order to keep the experimental system as simple as possible for confirming the role of MCP-1 in regulating the cytokine release by cells in response to oxidative stress. The addition of 100 μM H2O2 to mouse BV-2 microglial cells significantly enhanced their secretion of MCP-1 (Fig. 7A) and IL-4 (Fig. 7B), similar to the increases observed in the brains following VWR (Fig. 7A and 2D). Neutralizing MCP-1 by anti-murine MCP-1 antibodies significantly reduced the levels of not only MCP-1, but also IL-4 in the BV-2 cell supernatants. Thus, microglial IL-4 secretion could be modified by reducing extracellular levels of MCP-1. Secretion of TNF-α, IL-10 and IFN-γ by BV-2 cells was also studied, but no induction by 100 μM H2O2 was observed for these cytokines (data not shown).

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Fig. 7. Secretion of MCP-1 (A) and IL-4 (B) by BV-2 cells that were left untreated or stimulated

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with 100 μM H2O2 in the presence or absence of two concentrations of anti-murine MCP-1 antibody for 48 h. Data (means ± SEM) from 4 independent experiments are presented. The

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detection limits of the corresponding ELISAs are shown as dotted lines. a P < 0.05, compared to cells stimulated with H2O2 in the absence of antibody, according to the one-way ANOVA

are also shown.

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followed by Dunnett’s post-hoc test. P and F values for the main effect of the one-way ANOVA

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4. Discussion We chose to investigate the frontal cortex, hippocampal area and cerebellum since these

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areas of the brain respond to PA as well as to immune stimulation (Attwell et al., 2002; Basso et

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al., 2015; Bordner et al., 2011; Dishman et al., 2006; Murdoch et al., 2016; Rhodes et al., 2005;

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Wolf et al., 2009). Studies have shown that following acute exercise, neurons in the frontal cortex are more excitable, and cognitive abilities associated with frontal cortex processing are improved

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(Basso et al., 2015; Murdoch et al., 2016). Other studies have demonstrated that the age-related decline in motor performance correlates with increased markers of the CNS immune activation,

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including increased markers of complement activation and CD52 expression in the frontal cortex

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of mice (Bordner et al., 2011). The hippocampus, which is known for its role in learning and

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memory, has also been shown to be highly affected by PA. Exercise has been shown to increase the total number of hippocampal neurons and their activation, as well as the hippocampal production of GDNF, BDNF and several synaptic proteins such as synapsin 1, synaptophysin and postsynaptic density protein-95 (Aguiar et al., 2014; Erickson et al., 2011; Fernandes et al., 2016; Rhodes et al., 2003). Additionally, an increase in hippocampal neurogenesis in mice has been demonstrated following intraperitoneal administration of bacterial proteins, which clearly shows a hippocampal response to peripheral immune stimulation (Wolf et al., 2009). The cerebellum was an obvious choice for this study, since this brain region plays a critical role in motor movement and motor memory by both controlling and responding to PA (Attwell et al., 2002; De Zeeuw and Ten Brinke, 2015). Recent research highlights that moderate exercise and PA have potent anti-inflammatory

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ACCEPTED MANUSCRIPT activities in the periphery. However, very few studies have looked at the effects of PA on the immune status of the brain. We investigated CNS levels of several pro- and anti-inflammatory

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cytokines to establish the cytokine response in the brain following VWR. We showed that VWR induced an overall increase in pro-inflammatory (MCP-1 and TNF-α) and anti-inflammatory (IL-

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10 and IL-4) cytokines in the frontal cortex, hippocampus and cerebellum of MCP-1+/+ VWR

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mice. We propose that this overall increase in brain cytokine levels represents activation of the

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neuroimmune system in the VWR group, as opposed to the less activated immune system found in SED mice. An activated immune system is more proficient at recognizing and responding

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appropriately to endogenous stimuli and pathogenic invaders (Dilger and Johnson, 2008; Yeh et al., 2006). Animals were purposefully housed in the absence of a locked running wheel or toys to

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better model a SED environment and prevent mice from using objects placed in the cage for

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climbing and other forms of PA (Koteja et al., 1999). However, the lower level of environmental

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enrichment caused by the absence of running wheel in the cages of animals assigned to the SED group may have contributed to the observed difference in brain immune characteristics between animals from the PA and SED groups (Jurgens and Johnson, 2012). Additionally, this study was performed using female mice only; it is possible that the hormonal responses of these mice to VWR could represent an unknown confounding factor, and thus represents a limitation of this study (Fragala et al., 2011). Our data, showing that VWR leads to increases in select brain cytokines levels, are similar to previous findings of Nichol et al. (2008), who reported that Tg25765 mice models of Alzheimer’s disease had increased levels of IFN-γ and macrophage inflammatory protein (MIP)1α in the hippocampus following exercise (Nichol et al., 2008). However, our study revealed that VWR did not increase levels of brain IFN-γ, which is typically associated with a robust pro-

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ACCEPTED MANUSCRIPT inflammatory environment (Ishizuka et al., 2012; Schroder et al., 2004), thereby indicating that the increase in brain cytokine levels in MCP-1+/+ VWR mice does not necessarily represent a pro-

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inflammatory environment but could indicate glial cell activation making them more proficient at

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protecting the CNS (Dilger and Johnson, 2008).

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We also investigated the role that MCP-1 plays in mediating VWR-induced neuroimmune responses. We focused on MCP-1, since it is upregulated during inflammatory events in the CNS

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and has also been shown to regulate tight junctions at the BBB (Paul et al., 2014; Roberts et al.,

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2012), and is therefore critical in mediating cross talk between the peripheral and central immune systems. We showed that MCP-1-/- VWR mice displayed elevated brain levels of TNF- and IL-4

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compared to the MCP-1+/+ VWR group. However, MCP-1-/- VWR mice lost the ability to induce

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IL-10 expression compared the MCP-1+/+ VWR group. These data indicate that brain cytokine responses to VWR are at least partially regulated by MCP-1. In contrast to MCP-1+/+ VWR mice,

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MCP-1-/- VWR mice displayed a significant increase in IFN-γ, a powerful pro-inflammatory cytokine, indicating that lack of MCP-1 mediates a different inflammatory response in the brain. We cannot rule out that increased CNS infiltration of peripheral immune cells secreting various cytokines may have contributed to the observed changes in the CNS cytokine concentrations (Coisne et al., 2013). However, it is likely that these changes in brain cytokine levels in response to VWR were caused by the resident brain glial cells becoming activated in response to PA in an MCP-1-mediated manner. Activation of glial cells is critical for the maintenance of CNS homeostasis under normal physiological conditions, as well as during acute tissue damage and other pathological events. Astrocyte and microglial activation is most commonly measured as upregulated expression of GFAP and IBA-1 respectively (Sun et al., 2008); therefore, we used these proteins as indicators of glial cell activation. Astrocytes, but not 31

ACCEPTED MANUSCRIPT microglia became activated in response to VWR. These results are consistent with previous research, which indicated that PA enhances astrocyte activation (Saur et al., 2014). Other studies

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have demonstrated an overall decline in microglia activation following PA in aged mice (Kohman et al., 2012), as well as declined activation of both astrocytes and microglia in mouse models of

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Alzheimer’s disease upon introduction of PA (Leem et al., 2011; Nichol et al., 2008). A great deal

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of research has shown that glial cells behave differently with age (Lynch et al., 2010; Rozovsky et

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al., 1998) and in individuals with neurological diseases (Cameron and Landreth, 2010; Dheen et al., 2007; Reynolds et al., 2008); however, our study was specifically designed to investigate the

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neuroimmune-modifying effects of PA in non-aged, non-diseased animals to avoid these potential confounders. Since previous research has shown that PA reduces the chronic glial cell over-

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activation associated with age and neurodegenerative disease, and our data demonstrate that glial

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activation is enhanced in VWR mice compared to their SED counterparts, it could be suggested

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that regular/moderate PA maintains the activation of glia within a homeostatic range, thus achieving an overall beneficial effect. One interpretation of these data is that the neuroimmune responses to VWR may be contingent on MCP-1. A complementary interpretation is that MCP-1-/mice represent a different neuroimmune phenotype all together, since MCP-1-/- VWR mice had lower expression of IL-10, GFAP, IBA-1 and STAT1, but expressed higher levels of TNF-α and IFN- compared to MCP-1+/+ mice. Therefore, MCP-1 could be modulating the neuroimmune responses to VWR through direct or indirect mechanisms. It has been suggested that the immune-modulating effect of PA in the periphery is due to the downregulation of TLR4 expression on several cell and tissue types (Gleeson et al., 2011; McFarlin et al., 2006). TLR4 is primarily known to stimulate inflammation in response to bacterial lipopolysaccharide (LPS) (Gleeson et al., 2006). However, TLR4 triggers activation of

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ACCEPTED MANUSCRIPT inflammatory signaling cascades in response to a variety of other stimuli, such as heat shock proteins (HSP), high-mobility group box 1, fibrinogen and polyunsaturated fatty acids (Erridge,

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2010; Kilmartin and Reen, 2004; Lee et al., 2003; Park et al., 2004; Smiley et al., 2001). It has been proposed that elevated levels of HSP60, in particular, following PA may lead to cross-

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tolerance of TLR4, and subsequently result in the downregulation of TLR4 on the surface of

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several peripheral cell types (Kilmartin and Reen, 2004). We showed that there is a trend towards

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increasing expression of TLR4 in the frontal cortex of MCP-1+/+ VWR mice relative to MCP-1+/+ SED mice, demonstrating that the CNS response to PA may be different from what is observed in

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the periphery.

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In addition to the changes in TLR4 expression following PA, STAT1 is significantly and

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transiently upregulated in myocytes following exercise (Begue et al., 2013). STAT1 has been associated with infiltration of immune cells across the BBB (Chaudhuri et al., 2008) and total

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STAT1, as opposed to phosphorylated STAT1, is positively correlated with macrophage infiltration in breast cancer tumors (Tymoszuk et al., 2014). We hypothesized that STAT1 expression levels may increase in the brain following PA. However, we found that VWR had no effect on the expression of STAT1 in the frontal cortex of mice. We did discover, however, that STAT1 expression was significantly reduced in MCP-1-/- VWR mice; therefore, STAT1 may represent another key signaling molecule that is regulated by PA differentially in peripheral tissues compared to the CNS. It is interesting to note that MCP-1-/- mice had an overall reduction in neuroinflammation compared to the MCP-1+/+ mice, as evident by a decrease in glial cell activation and total STAT1 levels in the frontal cortex. We also showed that VWR induced a neuroimmune response, and MCP-1 appeared to partially govern these reactions. 33

ACCEPTED MANUSCRIPT Measurements of serum cytokine concentrations showed that neither VWR nor presence or absence of MCP-1 had an effect on serum levels of TNF-α, IL-10, IL-4 or IFN-γ. It could be

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proposed that the changes in CNS levels of cytokines can be independent from their peripheral concentrations. The observed lack of changes in peripheral cytokine levels in response to VWR

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differs from previous research (Fingerle-Rowson et al., 1998; Gleeson et al., 2011). This

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discrepancy could be due to the more intense exercise regimens used in most previous studies

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(Abbasi et al., 2014; Gleeson et al., 2011; McFarlin et al., 2004; Stewart et al., 2005). This discrepancy could also be due to the more intense exercise regimens used in most previous studies

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(Abbasi et al., 2014; Gleeson et al., 2011; McFarlin et al., 2004; Stewart et al., 2005). It is important to note that we did not perform a comprehensive evaluation of the peripheral immune

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status of the animals studied; therefore, even though serum cytokine levels did not change in these

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mice, we cannot rule out the possibility that PA stimulated other aspects of peripheral immune

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response, such as T cell production by the spleen, activation status of monocytes or adipose tissue macrophage phenotype (Gleeson et al., 2011). Since microglia represent a significant source of locally produced brain cytokines (Block et al., 2007; Dheen et al., 2007), these cells could be responding to VWR by altering their secretory profile in an MCP-1-dependent manner (Deshmane et al., 2009). We showed that by reducing MCP-1 concentration in cell culture medium, it was possible to reduce the secretion of IL-4 by BV-2 mouse microglial cells. These data support previous studies demonstrating that regulation of microglial cytokine secretion by MCP-1 is possible (Deshmane et al., 2009). However, the in vitro data did not match perfectly the effects that were observed in vivo, which is most likely due to the more complex nature of the CNS environment, including the presence of different cell types and the BBB.

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5. Conclusion Although the neuroimmune-modifying effects of exercise and PA are just emerging,

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studies indicate that PA has the ability to impact the immune system of the CNS. For example, PA

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can reduce over-activation of glial cells, such as during brain disease and old age (Cameron and

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Landreth, 2010; Dheen et al., 2007; Lynch et al., 2010; Reynolds et al., 2008; Rozovsky et al., 1998), and long-term exercise has been shown to reduce acute neuroinflammation resulting from

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traumatic brain injury (Piao et al., 2013). Clinical studies indicate that leading a physically active lifestyle can reduce the risk of developing Alzheimer’s disease by up to 2.7-fold (Barnes and

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Yaffe, 2011; Scarmeas et al., 2009), and Parkinson’s disease by approximately two fold (Zou et

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al., 2015), with other studies showing that resistance training can delay the progression of

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amyotrophic lateral sclerosis (Bello-Haas et al., 2007). Our data indicate that PA may induce astrocyte activation, enhance TLR4 expression and elevate levels of pro- and anti-inflammatory cytokines in the brain. Combined, these data indicate that the immune-modulating effect achieved by PA in the brain allows for modest, yet significant, increases in the expression of immune modifying receptors and cytokines, which ultimately may enable the immune system to mount a more rapid and effective response to endogenous or exogenous stimuli. In contrast, it is possible that in the absence of these effects, as observed in the SED group, the neuroimmune system lacks the ability to effectively respond to noxious stimuli; thus representing overall less activated immune system, as has been proposed by previous studies (Gleeson et al., 2011; Smith et al., 1996). Our finding that MCP-1 modulates the neuroimmunemodifying effects of VWR opens up research avenues for developing neuroprotective strategies

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their fundamental pathological features (Cameron and Landreth, 2010; Czlonkowska et al., 2002).

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Acknowledgements LS is supported by a Natural Sciences and Engineering Research Council of Canada

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(NSERC) scholarship and the UBC Okanagan Campus fellowships. ME is funded by an NSERC

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scholarship. DG is supported by a grant from NSERC. SG is supported by a Scholar Award from

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the Michael Smith Health Research Foundation and operating grants from the Canadian Diabetes Association, and the UBC Okanagan Campus. AK is supported by grants from the Jack Brown

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and Family Alzheimer’s Disease Research Foundation, NSERC and the UBC Okanagan Campus.

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ACCEPTED MANUSCRIPT Highlights Voluntary wheel running (VWR) was used to model physical activity in mice

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Increased levels of inflammatory cytokines were detected in the brains of VWR mice

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VWR enhanced astrocyte activation, but did not affect microglia activation

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Neuroimmune effects of VWR were explored in wild-type and MCP-1 knock out animals

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Neuroimmune responses to VWR were at least partially mediated by MCP-1

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