of June 28, 2018.This information is current as

Infiltration MacrophagehighSecretion and Gr1

Regeneration through Facilitating MCP-1 CD8 T Cells Are Involved in Skeletal Muscle

Wang and Jie DuJing Zhang, Zhicheng Xiao, Chao Qu, Wei Cui, Xiaonan

http://www.jimmunol.org/content/193/10/5149doi: 10.4049/jimmunol.1303486October 2014;

2014; 193:5149-5160; Prepublished online 22J Immunol 

Referenceshttp://www.jimmunol.org/content/193/10/5149.full#ref-list-1

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The Journal of Immunology

CD8 T Cells Are Involved in Skeletal Muscle Regenerationthrough Facilitating MCP-1 Secretion and Gr1high

Macrophage Infiltration

Jing Zhang,1 Zhicheng Xiao,1 Chao Qu, Wei Cui, Xiaonan Wang, and Jie Du

Inflammatory microenvironments play a key role in skeletal muscle regeneration. The infiltration of CD8 T cells into injuredmuscle

has been reported. However, the role of CD8 T cells during skeletal muscle regeneration remains unclear. In this study, we used

cardiotoxin-induced mouse skeletal muscle injury/regeneration model to investigate the role of CD8 T cells. Muscle regeneration

was impaired and matrix deposit was increased in CD8a-deficient mice compared with wild-type (WT) mice whose CD8 T cells

were infiltrated into damaged muscle after cardiotoxin injection. Adoptive transfer of CD8 T cells to CD8a-deficient mice

improved muscle regeneration and inhibited matrix remodeling. Compared with WT mice, CD8a deficiency limited the recruit-

ment of Gr1high macrophages (MPs) into muscle, resulting in the reduction of satellite cell number. The expression of MCP-1

(MCP-1/CCL2), which regulates the migration of Gr1high MPs, was reduced in CD8a-deficient mice compared with WT mice.

Coculture CD8 T cells with MPs promoted MCP-1 secretion. The i.m. injection of MCP-1 markedly promoted the recruitment of

Gr1high MPs and improved muscle regeneration in CD8a-deficient mice. We conclude that CD8 T cells are involved in skeletal

muscle regeneration by regulating the secretion of MCP-1 to recruit Gr1high MPs, which facilitate myoblast proliferation. The

Journal of Immunology, 2014, 193: 5149–5160.

Skeletal muscle injury induced by trauma, contraction,chemicals, ischemia, and neurologic dysfunctions is a rel-atively common injury (1). The injured skeletal muscle

has a remarkable capability to repair through regenerating newmuscle fibers. The main players participating in skeletal muscleregeneration are satellite cells, which lie beneath the muscle fiberbasal lamina, adjacent to the plasma membrane (2, 3). Duringmuscle regeneration, the quiescent satellite cells become activatedthrough asymmetric division. Some cells differentiate into myo-blasts, whereas others maintain stem cell characteristics and returnto quiescence (4, 5). These differentiated myoblasts migrate tocontact and fuse with each other to form myotubes. Newly formedmyotubes with central nucleation grow and further differentiateinto mature myofibers with nuclei moving to a subsarcolemmalposition (6, 7).The activation of satellite cells is a complex process that involves

interaction between satellite cells and the inflammatory microen-

vironment. As a part of the inflammatory microenvironment, themonocytes/macrophages (MOs/MPs) play an indispensable role in

skeletal muscle regeneration (8). Depletion of circulating MOs atthe injury time blocked muscle regeneration, whereas depletionof i.m. MPs at later stage prevented the differentiation of myo-blasts (9). During muscle regeneration, proinflammatory MOs were

recruited to the injury site and removed necrotic debris, followedby a switch from proinflammatory MOs into anti-inflammatoryMPs to promote myofiber growth (9). One important way to regu-late MP infiltration is through the secreting of chemokines, whichare named as C, CC, CXC, or CX3C chemokines according to

the number and location of the cysteine residues at N terminus(10). CCL2, also named as MCP-1, is expressed by many cells,including endothelial, fibroblasts, epithelial, smooth muscle, mesan-gial, astrocytic, monocytic, and microglial cells (11). The bio-logical effect of MCP-1 is mediated through interaction with its

receptor, CCR2, which is expressed on mononuclear cells, vas-cular smooth muscle cells, MOs, and activated NK cells (12).Deficiency of either CCL2 or CCR2 impaired muscle regeneration(13, 14). Furthermore, knockout (KO) of CXCL16 prevented MP

accumulation in injured muscle and blocked muscle regeneration(15). Although MOs/MPs are crucial for muscle regeneration, themolecular and cellular mechanisms that regulate MP recruitmentare still unclear.According to the surface markers, MPs can be classified into

different subsets, and different subsets correspond to different typesof functionality (16–18). The infiltration of different MP subsetshas been documented during skeletal muscle regeneration (9).Recently, experimental reports suggest that other immune cells,

such as T cells, also participate in the skeletal muscle regeneration.The absence of T lymphocytes in the animal model of dysferlin-opathy improved the muscle regeneration (19). Accumulation ofCD8 T cells has been documented in the mdx mouse model of

Duchenne muscular dystrophy (20). In the cardiotoxin (CTX)-induced skeletal muscle regeneration model, CD8 T cells werestill present in the muscle on day 14 after damage in Casitas

Beijing Anzhen Hospital, Capital Medical University, Beijing 100029, China; TheKey Laboratory of Remodeling-Related Cardiovascular Diseases, Capital MedicalUniversity, Ministry of Education, Beijing 100029, China; and Beijing Institute ofHeart, Lung and Blood Vessel Diseases, Beijing 100029, China

1These authors contributed equally to this work.

Received for publication January 2, 2014. Accepted for publication September 11,2014.

This work was supported by the National Natural Science Foundation of China(Grants 31090363, 81100144, 81430050, 81230006, 30888004, and 30971471) andthe Beijing Collaborative Innovative Research Center for Cardiovascular Diseases(PXM2014_014226_000002).

Address correspondence and reprint requests to Dr. Jie Du, Beijing Anzhen Hospital,Capital Medical University, 2 Anzhen Road, Chaoyang District, Beijing 100029, China.E-mail address: jdu@bcm.edu

Abbreviations used in this article: BMDM, bone marrow–derived macrophage; Cbl-b,Casitas B-lineage lymphoma-b; CD8 KO, CD8a-deficient; CD8 KO M, CD8 KO micewith MCP-1 administration; CD8 KO R, CD8 KO mice with CD8 T cell administration;CSA, cross-sectional area; CTX, cardiotoxin; KO, knockout; MO, monocyte; MP,macrophage; MPC, myogenic precursor cell; TA, tibialis anterior; WT, wild-type.

Copyright� 2014 by The American Association of Immunologists, Inc. 0022-1767/14/$16.00

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B-lineage lymphoma-b (Cbl-b)–deficient mice, and the suppres-sion in CD8 T cell infiltration resulted in improvement of the muscleregeneration in Cbl-b–deficient mice (21). However, the CD8 T cellresponse to skeletal muscle injury is not well characterized and itsrole in regulating inflammatory microenvironments remains unclear.In this study, we investigated the role of CD8 T cells in CTX-

induced skeletal muscle injury. We also characterized the crosstalk between CD8 T cells and MPs during muscle regeneration.Our results demonstrated that CD8 T cells are important for regulat-ing inflammatory microenvironment via amplification of chemo-attractant and recruitment of MPs in muscle regeneration.

Materials and MethodsAnimals

The CD8a-deficient (CD8 KO) mice were from The Jackson Laboratory(Bar Harbor, ME) and were crossed into C57BL/6J background, and thelittermates were used as controls. The Guide for the Care and Use ofLaboratory Animals (National Institutes of Health Publication No. 85-23,1996) was followed, and the study was approved by the Animal Care andUse Committee of Capital Medical University.

Muscle injury/regeneration model

Wild-type (WT) and CD8 KO mice were studied at 8–12 wk old. Themuscle regeneration model was performed as described previously (22). Inbrief, after anesthesia with pentobarbital sodium, tibialis anterior (TA)muscles were injected with 10 mM CTX (Sigma-Aldrich, St. Louis, MO)in saline (40 ml), whereas the group of Sham muscles was injected withsaline. At different time points after damage, mice were sacrificed and TAmuscles were mounted and frozen in isopentane chilled with liquid ni-trogen and stored at 280˚C.

Histochemical and immunohistochemical analyses

Serial, transverse cryosections (7 mm thick) of the midbelly region TAmuscles were stained with H&E, Masson trichrome. To calculate thecross-sectional area of individual myofibers, we incubated the musclesections with Ab against laminin (1:100; ZSGB-BIO, Beijing, China) at 4˚Covernight, then incubated with goat anti-mouse IgG labeled with AlexaFluor 488 (1:500; Invitrogen, Carlsbad, CA). Some muscle tissues werefixed in 4% paraformaldehyde, embedded in paraffin, and sectioned (5 mmthick). These muscle sections were incubated with Ab against Pax7 (1:100;Abcam, Cambridge, MA), then incubated with secondary Ab and detectedwith 3,3-diaminobenzidine for immunohistochemistry.

Images were captured by the Nikon microscope eclipse 90i (Nikon,Tokyo, Japan) and analyzed by a person blinded to treatment with the use ofNIS-Elements BR 3.1 software (Nikon). The matrix deposit of muscle wascalculated by the proportion of collagen-stained areas to total area. Formyofiber cross-sectional area measurements, 5 nonoverlapping areas ofeach section were digitally captured and .250 myofibers were calculatedin each sample. The distribution of fiber sizes was expressed as a per-centage of myofibers examined. The pax7+ cell density was counted fromat least six randomly captured fields.

To detect the location of CD8 T cells and MPs, we incubated the musclesections with Ab against CD68 (1:200; Abcam), CD8 (1:100; BD Bio-sciences, San Jose, CA) at 4˚C overnight, then incubated them with goatanti-rabbit IgG labeled with Alexa Fluor 555 (1:500; Invitrogen) and goatanti-rat IgG labeled with Alexa Fluor 488 (1:500; Invitrogen). Images werecaptured by a Leica TSC-SP5 laser-scanning confocal microscope (Leica,Wetzlar, Germany).

Flow cytometry

Flow cytometry was performed using muscle single-cell suspension, whichwas prepared as described with minor modifications (23). In brief, TAmuscles were dissected and then gently torn with tissue forceps. The firstround of enzymatic digestion was performed with Collagenase type II (2.5U/ml; Sigma-Aldrich) at 37˚C for 30 min. After washing, a second roundof enzymatic digestion was performed with Collagenase D (1.5 U/ml;Roche Diagnostics GmbH, Mannheim, Germany) and Dispase II (2.4 U/ml;Roche Diagnostics) at 37˚C for 30 min, and cells were collected at 300 3 gfor 10 min. Flow cytometry was carried out using the following Abs: FITCanti-mouse Gr1 (eBioscience, San Diego, CA), PE anti-mouse F4/80(Biolegend, San Diego, CA), PerCP-Cy5.5 anti-mouse CD45.2 (BDBiosciences), PE-CF594 anti-mouse CD3e (BD Biosciences), allophyco-cyanin anti-mouse CD4 (BD Biosciences), allophycocyanin-Cy7 anti-mouse

CD8a (BD Biosciences), allophycocyanin-Cy7 anti-mouse CD11b (BDBiosciences), FITC anti-mouse CD206 (Biolegend), and allophycocyaninanti-mouse Gr1 (BD Biosciences). Stained cells were analyzed by MoFlo(Beckman Coulter, Miami, FL), and data were collected using Summit 5.2software (Beckman Coulter). Cell sorting was performed on the MoFlo(Beckman Coulter). Debris and dead cells were excluded by forward scatter,side scatter, and DAPI gating.

Transfer of CD8 T cells into CD8a-deficient mice

T lymphocytes were isolated from the spleen of WT mice; then the CD8T cells were purified by Mouse CD8 Negative Isolation Kit (Invitrogen)according to the manufacturer’s instructions. The splenic CD8 T cellsisolated from WT mice (8–12 wk) were i.v. injected into CD8 KO mice(13 107 cells/mouse). Three hours later, the WT mice, CD8 KO mice, andCD8 KO mice with CD8 T cell administration (CD8 KO R) were injectedwith CTX in the TA muscles. Three days later, the group of CD8 KO Rwas i.v. injected with splenic CD8 T cells again (1 3 107 cells/mouse).Fourteen days later, the mice were sacrificed and the TA muscles werecollected for histochemical and immunohistochemical analyses.

RNA isolation and quantitative real-time PCR

The Gr1high and Gr1low MPs were isolated by cell sorting from skeletalmuscle at 2 d after CTX-induced injury. Total RNA of Gr1high or Gr1low

MPs was isolated with TRIzol reagent, according to the manufacturer’sprotocol (Invitrogen). Equal amounts of RNA (1 mg) were added to reversetranscriptase reaction mix with oligo-dT primers (Promega, Southampton,U.K.). SYBR Premix Ex Taq (TaKaRa, Shiga, Japan) was used to performquantitative real-time PCRs with IQ5 Multicolor Real-time PCR DetectionSystem (Bio-Rad, Hercules, CA). The following primers were used: CCR2(59-AGTTCATCCACGGCATACTATCAA-39 forward; 59-GCCCCTTCA-TCAAGCT-CTTG-39 reverse), Ly6C (59-AGACCCGTCAGTGCCTTTC-TT-39 forward; 59-CTGA-TGTTAGGATCCCTGATTGG-39 reverse), TNF-a(59-CACAAGATGCTGGGACAG-TGA-39 forward; 59-TCCTTGATGGT-GGTGCATGA-39 reverse), IL-1b (59-CCATGG-CACATTCTGTTCAAA-39 forward; 59-GCCCATCAGAGGCAAGGA-39 reverse), Arg1 (59-AA-CACGGCAGTGGCTTTAAC-39 forward; 59-GAGGAGAAGGCGTTT-GCTTA-39 reverse), IL-10 (59-CCAGGGAGATCCTTTGATGA-39 forward;59-CATT-CCCAGAGGAATTGCAT-39 reverse), and GAPDH (59-CATG-GCCTTCCGTGTTCCTA-39 forward; 59-GCGGCACGTCAGATCCA-39reverse).

Coculture of MPs and myoblasts

Noncontacting transwell cell culture system was prepared as follows:C2C12 myoblasts (1.5 3 104/well) were seeded on the bottom of 24-wellculture plate, and the Gr1high or Gr1low MPs (3 3 104/well) isolated byflow cytometry sorting were cultured onto the membrane of transwell cellculture inserts (pore size, 0.4 mm; Millipore, Billerica, MA) using DMEMhigh-glucose medium supplemented with 10% FBS and 1% penicillin-streptomycin. After coculture for 72 h at 37˚C with 5% CO2, the num-ber of C2C12 myoblasts was calculated by flow cytometry.

Cell migration assay

Cell migration was quantitated by use of 24-well Transwell inserts withpolycarbonate membrane filter (8-mm pore size; Corning, Corning, NY).The Gr1high or Gr1low MPs (1 3 104/well) isolated by cell sorting fromskeletal muscle at 2 d after CTX-induced injury were seeded onto theupper chamber of the insert. The bottom chambers were filled with 0.6 mlDMEM containing 1% FBS with recombinant murine MCP-1 (MCP-1/CCL2, 10 ng/ml; Peprotech, Rocky Hill, NJ) or recombinant humanfractalkine (CX3CL1, 10 ng/ml; Peprotech). Control groups contained 1%FBS without MCP-1 or CX3CL1 in bottom chambers. These MPs wereallowed to migrate for 12 h at 37˚C with 5% CO2.

MCP-1 concentration

The injured muscle MCP-1 concentration was determined by CBA FlexSet beads assay (BD Biosciences). The fluorescence produced by thebeads was measured on a FACSCalibur flow cytometer (BD Biosciences).

Coculture of MPs and T cells

Isolation of primary bone marrow–derived MPs (BMDMs) was performedas described previously (24). In brief, bone marrow cells were obtainedfrom the interface of PBS and Ficoll (HaoYang, TianJin, China) afterdensity centrifugation; then cells were resuspended and cultured in RPMI1640 medium (Life Technologies, Grand Island, NY) supplemented with10% FBS in the presence of 50 ng/ml recombinant murine M-CSF

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(Peprotech). WT CD8 or CD4 T cells were isolated from spleen andpurified by CD8 (Invitrogen) or CD4 cell isolation kit (Miltenyi Biotec,Auburn, CA) according to the manufacturer’s instructions. CD8 or CD4T cells (5 3 104/well) with or without BMDMs (5 3 104/well) were platedin 24-well plates and cultured in complete RPMI 1640 medium with anti-mouse CD3 and anti-mouse CD28 Abs (eBioscience) for 48 h. After incu-bation, 50 ml supernatant from each well was collected for the measurementof MCP-1 concentration by CBA Flex Set beads assay (BD Biosciences).

MCP-1 treatment before CTX injection

For MCP-1 treatment, the mouse received an injection of MCP-1 (50 ng/25ml/muscle) into the TA muscle. One hour later, the WT mice, CD8 KOmice, and CD8 KO mice with MCP-1 treatment (CD8 KO M) wereinjected with CTX in the TA muscles.

Statistical analysis

All data are expressed as mean6 SD. Statistics were calculated with SPSScomputer software for Windows (version 13.0; SPSS, Chicago, IL). Stu-dent t test was used to compare data between two groups in each experi-ment. One-way ANOVA, followed by a Bonferroni–Dunn test, was used tocompare data between three or more groups in each experiment. Resultswere considered to represent significant differences at p , 0.05.

ResultsCD8 T cells infiltrate into injured skeletal muscle

To investigate the impact of T cells in skeletal muscle regeneration,we injected CTX into TA muscles to induce the muscle injury/regeneration. The injured muscles were digested into cell sus-pension at different time points and analyzed by flow cytometry. InWT mice, the peak infiltration of both CD4 and CD8 T cells wasdetected at day 3 and gradually decreased thereafter; both CD4 andCD8 T cells were hardly detected at day 14 (Fig. 1A). At day 3,CD3+CD4+ T cells were 1.1 6 0.2% and CD3+CD8+ T cells were0.9 6 0.1% of stromal cells in injured skeletal muscle tissue(Fig. 1B, 1C). Immunofluorescence staining showed the presenceof CD8 expression in the injured skeletal muscle tissue at day 3.The CD8 T cells were located in the interstitial space; some ofthem were located adjacent to the regenerating myofiber (Fig. 1D).Thus, both CD4 and CD8 T cells are present in injured skeletalmuscle during muscle injury/regeneration.

CD8 T cells are involved in skeletal muscle regeneration

To determine the role of CD8 T cells in skeletal muscle regenera-tion, we used CD8 KOmice that are deficient in functional cytotoxicT cells, whereas Th cell development and function is normal (25).CTX was injected into TA muscles of WT and CD8 KO mice, re-spectively. Fourteen days after CTX injection, H&E-stained musclesections showed that newly formed myofibers (indicated by centralnuclei) of CD8 KO mice were smaller than WT mice (Fig. 2A).The mean muscle fiber cross-sectional area (CSA; mM2) of CD8KO mice was poor (896.06 47.0), compared with that of WT mice(1215.7 6 159.5, p , 0.05, Fig. 2B). Muscle regeneration was alsoquantified by the ratio of TA muscle weight to body weight (&)after regeneration (day 7: 1.5 6 0.1 in WT mice, 1.2 6 0.1 in CD8KO mice, p , 0.01; day 14: 1.9 6 0.1 in WT mice, 1.6 6 0.1 inCD8 KO mice, p , 0.05; Fig. 2C). The size distribution of myo-fibers showed fiber sizes have a significantly left shift in CD8 KOmice from that in WT mice, which indicated that muscle regener-ation capacity was decreased in CD8 KO mice (Fig. 2D–F). We alsodetected matrix deposit in injury/regenerating muscle. Fourteendays after injury, collagen deposition was measured by Massontrichrome staining. The regenerating muscle of CD8 KO miceshowed significantly more matrix deposit than that of WT mice,whereas no matrix deposit was found in uninjured muscle in bothWT and CD8 KO mice (Fig. 2G, 2H). Therefore, CD8a deficiencyinhibits regeneration and promotes matrix remodeling during skel-etal muscle regeneration.

Transfer of CD8 T cells into CD8 KO mice rescues theimpaired muscle regeneration

To examine the role of CD8 T cells in skeletal muscle regeneration,we adoptively transferred splenic CD8 T cells into CD8 KO mice.The splenic CD8 T cells were isolated from WT mice and i.v.administered into CD8 KO mice at 0 and 3 d after CTX injury. Theregeneration of skeletal muscle was analyzed at day 14. H&E-stained muscle sections showed that the newly formed myofibersin CD8 KO R were larger than that in CD8 KO mice (Fig. 3A). Themean muscle fiber CSA (mM2) was increased in CD8 KO R mice(1280.06 65.0) compared with that in CD8 KO mice (986.76 82.1,p , 0.01; Fig. 3B). The ratio of TA muscle weight to body weight(&) was 1.8 6 0.1 in WT mice, 1.6 6 0.1 in CD8 KO mice, and1.86 0.1 in CD8 KO R mice (Fig. 3C). The size distribution of myo-fibers was left shifted in CD8 KO mice and was virtually returnedto the levels of WT mice in CD8 KO R mice (Fig. 3D, 3E). Thematrix remodeling in regenerating muscle of CD8 KO R mice wasinhibited (6.06 1.3) compared with that in CD8 KOmice (13.86 3.4,p , 0.01; Fig. 3F, 3G). Taken together, these data indicate that thelack of CD8 T cell recruitment in injured muscle is responsiblefor the impaired muscle regeneration in CD8a-deficient mice.

CD8 T cells are involved in muscle recruitment of Gr1high MPsand proliferation of satellite cells

MP plays a key role during skeletal muscle regeneration, becausemuscle regeneration is impaired in the absence of MPs (9). Con-sidering the importance of MPs, we addressed whether infil-tration of MPs was regulated by CD8a deficiency. We measured i.m.MPs by flow cytometry. The F4/80 and CD11b Abs were used toidentify MPs, and the Gr1 Ab was used to identify different MPsubsets. After injured for 1 d, there were two subsets of F4/80+

CD11b+ MP accumulation in skeletal muscle. The subset withhigh Gr1 expression was named Gr1high, the other subset withlower Gr1 expression was named Gr1low (Fig. 4A). The percent-age of Gr1high MPs was higher in WT mice (23.6 6 0.4% ofCD45+ cells) compared with that in CD8 KO mice (17.6 6 2.0%of CD45+ cells, p , 0.01; Fig. 4B). There was no significantdifference in the infiltration of Gr1low MPs between CD8 KO andWT mice (Fig. 4C). The percentage of total MPs in WT mice(33.8 6 1.6% of CD45+ cells) was higher than that in CD8 KOmice (26.9 6 2.5% of CD45+ cells, p , 0.01; Fig. 4D), probablybecause of the impaired infiltration of Gr1high MP in CD8 KOmice. Therefore, CD8a deficiency limits muscle recruitment ofGr1high MPs in response to acute muscle injury.MPs can be broadly classified into M1 (classical) and M2

(alternative) subtypes based on the expression of CD206, which isan M2 MP surface marker. To clarify the relation between theGr1high and Gr1low populations and the M1 and M2 populations,we detected the expression of CD206 in Gr1high and Gr1low MPsby flow cytometry. A total of 63.3 6 1.2% of the Gr1low MPs wasCD206+, and 16.5 6 0.4% of the Gr1high MPs was CD206+

(Fig. 4E). These results indicated that Gr1low MPs were moresimilar to M2 MPs, and Gr1high MPs were more similar to M1 MPs.To examine the role of Gr1high MPs during muscle regeneration, weisolated both subsets from injured muscle by cell sorting (Fig. 4F).We analyzed gene expression of isolated Gr1high and Gr1low MPs byreal-time PCR. Results showed that Gr1high MPs expressed CCR2,Ly6C, TNF-a, and IL-1b more strongly than Gr1low MPs. Con-versely, Gr1low MPs expressed Arg1 and IL-10 more strongly thanGr1high MPs (Fig. 4G). These data combined with CD206 expressiondata demonstrated that Gr1high MPs would be considered as M1-likeMPs and Gr1low MPs would be considered as M2-like MPs.Coculture of C2C12 myoblasts with Gr1high or Gr1low different

subset MPs was performed to evaluate MP function. We used the

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noncontacting transwell coculture system to allow bidirectionaldiffusion of soluble factors. After 72 h, the number of C2C12myoblasts was calculated by flow cytometry. The number ofC2C12 myoblasts was increased in coculture with Gr1high MPs,but not with Gr1low MPs (Fig. 4H), which indicates that Gr1high

MPs promoted proliferation of C2C12 myoblasts.Considering Gr1high MPs promoted myoblast proliferation

in vitro, we addressed whether the decrease of Gr1high MPs result

in the reduction of satellite cell number in injured muscle. Fourdays after injury, the histological analysis of pax7 (satellite cellmarker) revealed that the number of pax7+ cells in WT mice wasmuch higher than that in CD8 KO mice (Fig. 4I, 4J). Taken to-gether, we found that CD8 T cells are involved in muscle recruit-ment of Gr1high MPs, which promote myoblast proliferation. Wealso demonstrate that the decrease of Gr1high MP is accompaniedwith reduction of satellite cells in injured muscle of CD8 KO mice.

FIGURE 1. CD8 T cells infiltrated into injured skeletal muscle. (A) Flow cytometry analysis of CD4 and CD8 T cells present in skeletal muscle at

different time points after CTX-induced injury. Representative examples of FACS analysis at each time point. (B) The cell population of CD4 T cells was

analyzed (n = 4 in each time point). Results are expressed as the percentage of total number of viable muscle interstitial cells. (C) The cell population of

CD8 T cells was analyzed (n = 4 in each time point). (D) Immunohistochemical analysis of CD8 in skeletal muscle at day 3 after CTX-induced injury. Top

panels present the low-magnification images of CD8 (red) and DAPI (blue). BF, brightfield image. White arrows indicate CD8+ cells; white arrowhead

indicates regenerating myofiber with central nuclei. Scale bar, 50 mm. Bottom panels present the high-magnification insets from the white boxes of the top

panels. Scale bar, 10 mm.

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Interaction between MPs and CD8 T cells stimulates MCP-1secretion, which facilitates Gr1high MP migration

To address whether reduced Gr1high MPs is related with decreasedchemokine secretion, we measured the production of MCP-1

(MCP-1/CCL2), which is one of the key chemokines that regu-

late migration and infiltration of MOs/MPs. The concentration of

MCP-1 was lower in the injured muscle of CD8 KO mice than

that of WT mice at 1 and 2 d after CTX injection (Fig. 5A). The

migration assay was performed to evaluate the chemotaxis of Gr1high

or Gr1low different subset MPs to MCP-1. MCP-1 directly promoted

migration of Gr1high MPs, but not Gr1low MPs (Fig. 5B), whereas

CX3CL1 did not affect the migration of both subsets of MPs.

After injury in WT mice, the immunofluorescence staining ofboth CD8 and MP marker CD68 demonstrated that CD8 T cellswere adjacent to MPs in skeletal muscle tissue (Fig. 5C). To ad-dress the interplay between CD8 T cells and MPs, we isolatedprimary splenic CD8 T cells, splenic CD4 T cells, and BMDMs,and cultured these cells, respectively, or cocultured BMDMswith either CD8 T cells or CD4 T cells. The conditioned mediumwas collected for detecting the MCP-1 concentration. The MCP-1was sharply increased in the cocultured CD8 T cells with BMDMs

compared with other groups (Fig. 5D).To determine the impact of MCP-1 on Gr1high MP in vivo, we

injected MCP-1 into the TA muscle of CD8 KO mice 1 h before

CTX injection. One day later, we measured the number of i.m.

FIGURE 2. CD8a deficiency inhibi-

ted regeneration and promoted matrix

remodeling. (A) H&E-stained muscles

from WT and CD8 KO mice were ex-

amined at 14 d after injury. Scale bar,

50mm. (B) Thegraph indicated themean

CSA of muscle myofiber in each group.

(C) Quantitative analysis showed that the

ratioofTAmuscleweight tobodyweight

(&) in each group at different time

points. (D) Muscles from WT and CD8

KO mice were immunostained with

laminin at 14 d after injury. Scale bar,

100 mm. (E and F) Graphs indicated the

distribution of myofiber sizes in WT (E)

and CD8 KO (F) mice. (G) Masson tri-

chrome–stained muscles from WT and

CD8 KO mice were examined at 14 d

after injury. Scale bar, 50 mm. (H)

Quantitative analysis showed the matrix

deposit (%) in each group. n = 4 mice/

group/time point. *p,0.05, **p,0.01.

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MPs by flow cytometry. A single injection of MCP-1 remarkably

improved muscle recruitment of Gr1high MPs, and the muscle

recruitment of total MP was also improved in CD8 KO mice

(Fig. 5E, 5F). Thus, CD8 T cells are important for the secretion of

MCP-1, which facilitates muscle recruitment of Gr1high MPs.

Local MCP-1 injection improves muscle regeneration in CD8KO mice

To address the role of MCP-1 in skeletal muscle regeneration ofCD8 KO mice, we injected MCP-1 into the TA muscle of CD8 KOmice 1 h before CTX injection. Fourteen days later, H&E-stained

muscle sections revealed that newly formed myofibers in CD8 KO

M was larger than that in CD8 KO mice (Fig. 6A). The mean

muscle fiber CSA (mM2) was increased in CD8 KO M (1326.3 678.4) compared with that in CD8 KO mice (924.06 87.9, p, 0.01;

Fig. 6B). The ratio of TA muscle weight to body weight (&) was

1.96 0.1 in WT mice, 1.66 0.1 in CD8 KO mice, and 1.96 0.1 in

CD8 KO M (Fig. 6C). The size distribution of myofibers was left

shifted in CD8 KO mice and was virtually returned to the levels ofWT mice in CD8 KO M (Fig. 6D, 6E). The matrix remodeling inregenerating muscle of CD8 KO M was inhibited compared withthat of CD8 KO mice (Fig. 6F, 6G). Therefore, elevated MCP-1 in

FIGURE 3. Transfer of CD8 T cells into CD8

KO mice rescued the impaired muscle regen-

eration. (A) H&E-stained muscles from WT

mice, CD8 KO mice, and CD8 KO mice i.v.

injected with CD8 T cells (CD8 KO R) were

examined at 14 d after injury. Scale bar, 50 mm.

(B) The graph indicated the mean CSA of

muscle myofiber in each group. (C) Quantitative

analysis showed the ratio of TA muscle weight

to body weight (&) in each group at 14 d after

injury. (D) Muscles from WT mice, CD8 KO

mice, and CD8 KO R were immunostained with

laminin at 14 d after injury. Scale bar, 100 mm.

(E) The graph indicated the distribution of

myofiber sizes in WT mice, CD8 KO mice, and

CD8 KO R. (F) Masson trichrome–stained

muscles from WT mice, CD8 KO mice, and

CD8 KO R were examined at 14 d after injury.

Scale bar, 50 mm. (G) Quantitative analysis

showed the matrix deposit (%) in each group.

n = 4–6 mice/group. *p , 0.05, **p , 0.01.

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FIGURE 4. CD8a deficiency blocked Gr1high MP recruitment and impaired satellite cell proliferation. (A) Flow cytometry analysis of Gr1high and Gr1low

MPs present in skeletal muscle of WT and CD8 KO mice at 1 d after CTX-induced injury. (B–D) The cell population of Gr1high (B), Gr1low (C), and total (D)

MPs was analyzed (n = 3 in each group). Results are expressed as the percentage of CD45+ cells isolated from muscle. (E) Flow cytometry analysis of F4/80 and

CD206 expression of Gr1high (down) and Gr1low (up) MPs at 2 d after CTX-induced injury (n = 4 in each group). (F) Gr1high and Gr1low MPs were isolated by

cell sorting from skeletal muscle at 2 d after CTX-induced injury. (G) Expression of CCR2, Ly6C, TNF-a, IL-1b, Arg1, and IL-10 was analyzed by real-time

PCR in isolated Gr1high and Gr1low MPs at 2 d after injury. GAPDH was used to normalize the quantitative real-time data. Results are expressed as relative fold

changes compared with Gr1low MPs (n = 4 in each group). (H) C2C12 myoblasts were cocultured with Gr1high or Gr1low MPs and analyzed for their pro-

liferation. (I) Immunohistochemical staining of Pax7 in skeletal muscle of WT and CD8 KO mice at 4 d after CTX-induced injury. Scale bar, 50 mm. (J)

Quantitative analysis of Pax7+ cell density, which is expressed as the number of Pax7+ cells per area (mm2). n = 4 mice/group. *p , 0.05, **p , 0.01.

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local injured muscle promotes regeneration in CD8 KO mice andCD8 T cells are involved in skeletal muscle regeneration (Fig. 7).

DiscussionHow inflammatory microenvironment is regulated during skeletalmuscle regeneration is not well characterized. In this study, weprovided evidence that CD8 T cells are involved in skeletal muscle

regeneration in response to CTX-induced muscle injury. We re-vealed a functional role of CD8 T cells in regulating inflammation

in muscle injury/regeneration process. CD8 T cells that interact

with MPs promote the MCP-1 secretion, resulting in recruitment of

Gr1high MPs, leading to myoblast proliferation in injured skeletal

muscle, and finally promote skeletal muscle regeneration.

FIGURE 5. Interaction between MP and CD8 T cells stimulated MCP-1 secretion, which facilitated Gr1high MP migration. (A) MCP-1 protein levels in

muscles of WT mice and CD8 KO mice were detected at 1 and 2 d after CTX-induced injury. n = 5–6 mice/group. (B) MCP-1 or CX3CL1 chemotaxis for

each MP subset was studied by Transwell with MCP-1 protein, CX3CL1 protein, or without protein (control) in the bottom chamber. The MPs were seeded

on the upper chamber. n = 4 in each group. (C) Immunohistochemical analysis of CD8 and CD68 in skeletal muscle of WT mice on 3 d after CTX-induced

injury. Scale bar, 5mm. (D) Concentration of MCP-1 in the condition medium of CD4 T cells (CD4+ T), CD8 T cells (CD8+ T), MPs (Mw), CD4 T cells

cocultured with MPs (CD4+ T with Mw), and CD8 T cells cocultured with MPs (CD8+ T with Mw). n = 4 in each group. **p , 0.01 versus all the other

groups. (E) Flow cytometry analysis of Gr1high and Gr1low MPs present in skeletal muscle of WT mice, CD8 KO mice, and CD8 KO M at 1 d after CTX-

induced injury. (F) The cell population of Gr1high, Gr1low, and total MPs was analyzed (n = 3 in each group). *p , 0.05, **p , 0.01.

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Skeletal muscle regeneration can be divided into three periods: thedestruction period characterized by the destruction of myofibers andthe accumulation of inflammatory cells; the regeneration pe-riod characterized by the activation, proliferation of satellite cells,and generation of new myofibers; and the remodeling periodcharacterized by the growth of newly formed myofibers and theremodeling of extracellular matrix (1). The coordinated action ofinflammatory cells and inflammatory factors during inflammatoryperiod is very important for the repair of the skeletal muscle.When muscle is injured, neutrophils are the first-response in-flammatory cells, which appear within 2 h in damaged muscle(10). In CTX-induced muscle regeneration, after the infiltration of

neutrophils, MPs begin to infiltrate, which increase and reach thepeak numbers in the muscle at ∼3 d, and gradually decreasethereafter. There is only a small number of MPs on day 7 (26). Asan important part of the inflammatory microenvironment, MPplays a key role in muscle regeneration, and its function has beenwidely recognized. In vivo, the suppression of MPs infiltrationleads to incomplete skeletal muscle regeneration and causesadipogenesis and fibrosis (27), whereas depletion of the MPsalso reduces the diameter of regenerating myofibers (28). In acuteskeletal muscle injury, the function of MPs is important. Theperiphery MO/MP depletion is associated with impaired repairafter acute skeletal muscle injury (9, 29). In the CTX injection–

FIGURE 6. Local MCP-1 injection improved

muscle regeneration in CD8 KO mice. (A) H&E-

stained muscles from WT mice, CD8 KO mice,

and CD8 KO mice i.m. injected with MCP-1

(CD8 KO M) were examined at 14 d after injury.

Scale bar, 50 mm. (B) The graph indicated the

mean CSA of muscle myofiber in each group. (C)

Quantitative analysis showed the ratio of TA

muscle weight to body weight (&) in each group

at 14 d after injury. (D) Muscles from WT mice,

CD8 KO mice, and CD8 KO M were immu-

nostained with laminin at 14 d after injury. Scale

bar, 100 mm. (E) The graph indicated the distri-

bution of myofiber sizes in WT mice, CD8 KO

mice, and CD8 KO M. (F) Masson trichrome–

stained muscles from WT mice, CD8 KO mice,

and CD8 KO M were examined at 14 d after

injury. Scale bar, 50 mm. (G) Quantitative anal-

ysis showed the matrix deposit (%) in each

group. n = 4 mice/group. *p , 0.05, **p , 0.01.

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induced acute muscle injury/regeneration model, we demonstratedthat CD8 T cell deficiency impaired muscle regeneration, and thetotal MPs and one subset of MPs (Gr1high) were suppressed in theinjured muscle of CD8 KO mice (Fig. 4A–D). Thus, in acuteskeletal muscle injury/regeneration, CD8 T cells are critical forregulating inflammatory microenvironment through control of theinfiltration of MPs.MPs can be divided into several subsets; different subsets of

MPs correspond to different function in skeletal muscle injury/regeneration. The MPs are classified into M1 (classical) and M2(alternative) subtypes. At early stage of skeletal muscle injury,M1 MPs invade into injured muscle. M1 MPs are characterizedby expressing proinflammatory cytokines TNF-a and IL-1b andothers (30). After M1 MPs reach their peak number in muscle,they are replaced by M2 MPs, which attenuate the inflammatoryresponse. M2 MPs are characterized by expressing CD206, Arg1,and anti-inflammatory cytokine IL-10, among others (31). In ourresearch, we identified two subsets of MPs, Gr1high and Gr1low,that were infiltrated into injured skeletal muscle. Gr1high MPsexpressed high levels of TNF-a and IL-1b, whereas Gr1low MPsexpressed high levels of CD206, Arg1, and IL-10 (Fig. 4E, 4G).Thus, the Gr1high MPs are phenotypically similar to classicallyactivated M1 MPs, and Gr1low MPs share features with alterna-tively activated M2 MPs. In the blood, circulating MOs can beclassified into at least two populations, CCR2+Ly6C+ MOs andCCR22y6C2 MOs. In the experimentally induced inflammation,CCR2+Ly6C+ MOs migrate into inflamed tissue and differentiateinto MPs and dendritic cells. In the absence of inflammation,CCR22Ly6C2 MOs enter the tissue and replenish the tissue-resident MPs and dendritic cells (32). In our study, Gr1high MPsexpressed high level of CCR2, Gr1low MPs expressed low level ofCCR2 (Fig. 4G), and both Gr1high and Gr1low MPs expressedLy6C (also known as Gr1; Fig. 4A). Previous research has dem-onstrated that the muscle recruited proinflammatory (M1) MPsfirst after injured; then these MPs switched their phenotype tobecome anti-inflammatory (M2) MPs through phagocytosis ofmuscle cell debris (9). Then we speculated that both Gr1high andGr1low MPs in muscle are derived from CCR2+Ly6C+ MOs inblood, and CCR2+Ly6C+ MOs first migrate into injured muscleand differentiate into Gr1high MPs, which switch into Gr1low MPsin muscle at a later time. In acute muscle injury research, twotypes of MOs/MPs, CX3CR1lo/Ly-6C+ and CX3CR1hi/Ly-6C2, areaccumulated in injured skeletal muscle during regeneration inCX3CR1GFP/+ mice. In vitro, the proinflammatory MOs/MPs in-

crease myogenic precursor cell (MPC) growth and proliferation,and the anti-inflammatory MOs/MPs promote MPC differentiationand fusion (9). In this study, the Gr1high MPs, but not Gr1low MPs,could promote the proliferation of satellite cells in vitro (Fig. 4H),and the decrease of Gr1high MPs related to the reduction of thenumber of satellite cells in vivo (Fig. 4I, 4J). These data demon-strated different effects of Gr1high and Gr1low MPs on muscleregeneration.The activation and proliferation of satellite cells play a key

role in muscle repair. Although recent findings demonstratedthat several other cell types (33–35) participated in muscle regen-eration, satellite cells are still recognized as the primary source forpostnatal skeletal muscle regeneration (36). After ablation ofsatellite cells, the muscle repair is prevented, and regenerated TAmuscle is completely lost 56 d postinjury (37). Previous studieshave indicated that Pax7, which is essential for satellite cellspecification and survival (38), is maintained in both quiescent andactivated satellite cells in adult mouse muscle (39), and the in-activation of Pax7 leads to complete ablation of satellite cells (40),and inhibits skeletal muscle regeneration (37, 41). Thus, satellitecells with Pax7 expression are indispensable for skeletal muscleregeneration (36). In CD8 KO mice, we observed less Pax7+

satellite cells compared with that in WT mice 4 d after injury(Fig. 4I, 4J), suggesting the deficiency in CD8 suppressed pro-liferation of satellite cells. According to the pivotal role of satellitecells for injured muscle regeneration, inhibition of satellite cellproliferation may be an underlying reason for impaired repair inCD8 KO mice. We also found that Gr1high MPs could promotesatellite cell proliferation independent of direct contacts in vitro(Fig. 4H). It might be related to the fact that MPs could influ-ence satellite cells through secretion of diverse growth factors andcytokines (28, 42). Previous study has shown that conditionedmedium from human MOs/MPs promotes human MPC prolifer-ation by soluble factors, and MOs/MPs facilitate MPC survival bydirect contacts (42). In our study, Gr1high MPs facilitate satellitecell proliferation through the secretion of factors, indicating theinfiltration of Gr1high MPs play a crucial role in muscle repair.MP infiltration is regulated by chemokines, which are small,

soluble molecules and can be classified into different subsets (10,11). Several studies have demonstrated strong evidence thatCC chemokines, especially CCL2/MCP-1, play an important rolein muscle regeneration. After injection with anti-CCL2, whichblocks the interaction with its receptor CCR2, the mice addressedpoor functional recovery (43). Disruption of CCL2/CCR2 sig-

FIGURE 7. A model of the role of CD8 T cells on

the processes of injured skeletal muscle regeneration.

Upon skeletal muscle injury, MOs and CD8 T cells

extravasate shortly from the blood vessel, then migrate

into the injured site. There is a reciprocal interaction

between infiltrated CD8 T cells and MPs, which stim-

ulates the cascade amplification of MCP-1. MCP-1

leads to the recruitment of Gr1high MPs, which promote

the proliferation of myoblasts and finally promote

muscle regeneration.

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naling showed markedly reduced invasion of MPs into the injurysite and impaired repair of acute skeletal muscle injury (13, 14,

44). The transplantation of bone marrow from WT mice restored

the muscle inflammation and reversed the defects in muscle re-

generation of CCR22/2 mice (45). CCL2/CCR2 signaling is re-

quired for recruitment of MOs/MPs, but not lymphocytes or

neutrophils, during skeletal muscle regeneration, and the mobili-

zation of MOs/MPs conducts phagocytosis and produces insulin-

like growth factor 1 to promote skeletal muscle repair. The

injection of insulin-like growth factor 1 into the injured muscle

facilitates muscle regeneration in both CCL22/2 and CCR22/2

mice (13, 14). Our results demonstrated that CD8 T cells are

important for production of MCP-1 in muscle regeneration. CD8

deficiency suppressed the production of MCP-1 after injury, and

MCP-1 guided the migration of Gr1high MPs (Fig. 5A, 5B). The

injection of MCP-1 into the injury muscle improved the accu-

mulation of Gr1high MPs, which promoted myoblast prolifera-

tion. The i.m. injection of MCP-1 also restored the regeneration

defects and inhibited the matrix remodeling in skeletal muscle

of CD8 KO mice (Fig. 6). Our findings suggest that CD8 T cells

regulate muscle regeneration via regulation of Gr1high MPs

attracted by MCP-1.To our knowledge, only rarely have reports demonstrated the

infiltration of CD8 T cells in injured muscle and the functional

role of CD8 T cell in muscle tissue inflammation. Wehling-

Henricks et al. (20) have reported that either ablation of ma-

jor basic protein-1 or eosinophil depletion induces increases

in CD8 T cells in mdx mice, which is the model of Duchenne

muscular dystrophy, a progressive and genetic disorder of

muscle degeneration. They found the accumulation of CD8

T cells in mdx mice, but did not study the functional role of

CD8 T cells in mdx mice. Kohno et al. (21) have demonstrated

that in CTX-induced acute muscle injury, CD8 T cells were still

present in the muscle on day 14 of Cbl-b–deficient mice, and

the suppression of CD8 T cell infiltration promoted the muscle

regeneration of Cbl-b–deficient mice. Kohno et al. (21) showed

that CD8 T cells seemed to impair skeletal muscle regenera-

tion, whereas our results indicate that CD8 T cells were im-

portant for skeletal muscle regeneration. The explanation of the

difference may be the different types of mice used for muscle

regeneration researches. In our study, we used WT and CD8a2/2

mice, and addressed that after CTX injection for 14 d, the CD8

T cells were hardly detected in WT mice. However, Kohno

et al. (21) used WT and Cbl-b2/2 mice, and showed that CD8

T cells could still be detected on day 14 after CTX injection in

Cbl-b2/2 mice, although they demonstrated no CD8 T cell

detection on day 14 after CTX injection in WT mice. In ad-

dition, different methods are used for researching the role of

CD8 T cells in muscle regeneration. Kohno et al. (21) used

RANTES neutralizing Ab to suppress the infiltration of CD8

T cells, whereas we used CD8a2/2 mice, which are deficient in

functional cytotoxic T cells. We thought CD8a2/2 mice may

be more specific to study the effects of CD8 T cells on muscle

regeneration. Meanwhile, Kohno et al. (21) also did not

show the role of CD8 T cell in regulation of muscle tissue

inflammation.In conclusion, our results have addressed the critical role for

CD8 T cells in regulating skeletal muscle regeneration (Fig. 7).

After acute muscle injury, the interaction between CD8 T cells

and MPs promotes the secretion of MCP-1 to guide the mi-

gration of Gr1high MPs, leads to myoblast proliferation in in-

jured skeletal muscle, and finally promotes skeletal muscle

regeneration.

DisclosuresThe authors have no financial conflicts of interest.

References1. Turner, N. J., and S. F. Badylak. 2012. Regeneration of skeletal muscle. Cell

Tissue Res. 347: 759–774.2. Tedesco, F. S., A. Dellavalle, J. Diaz-Manera, G. Messina, and G. Cossu. 2010.

Repairing skeletal muscle: regenerative potential of skeletal muscle stem cells. J.Clin. Invest. 120: 11–19.

3. Mauro, A. 1961. Satellite cell of skeletal muscle fibers. J. Biophys. Biochem.Cytol. 9: 493–495.

4. Price, F. D., K. Kuroda, and M. A. Rudnicki. 2007. Stem cell based therapies totreat muscular dystrophy. Biochim. Biophys. Acta 1772: 272–283.

5. Dhawan, J., and T. A. Rando. 2005. Stem cells in postnatal myogenesis: mo-lecular mechanisms of satellite cell quiescence, activation and replenishment.Trends Cell Biol. 15: 666–673.

6. Ten Broek, R. W., S. Grefte, and J. W. Von den Hoff. 2010. Regulatory factorsand cell populations involved in skeletal muscle regeneration. J. Cell. Physiol.224: 7–16.

7. Jarvinen, T. A., T. L. Jarvinen, M. Kaariainen, H. Kalimo, and M. Jarvinen.2005. Muscle injuries: biology and treatment. Am. J. Sports Med. 33: 745–764.

8. Pannerec, A., G. Marazzi, and D. Sassoon. 2012. Stem cells in the hood: theskeletal muscle niche. Trends Mol. Med. 18: 599–606.

9. Arnold, L., A. Henry, F. Poron, Y. Baba-Amer, N. van Rooijen, A. Plonquet,R. K. Gherardi, and B. Chazaud. 2007. Inflammatory monocytes recruited afterskeletal muscle injury switch into antiinflammatory macrophages to supportmyogenesis. J. Exp. Med. 204: 1057–1069.

10. Tidball, J. G., and S. A. Villalta. 2010. Regulatory interactions between muscleand the immune system during muscle regeneration. Am. J. Physiol. Regul.Integr. Comp. Physiol. 298: R1173–R1187.

11. Deshmane, S. L., S. Kremlev, S. Amini, and B. E. Sawaya. 2009. Monocytechemoattractant protein-1 (MCP-1): an overview. J Interferon Cytokine Res. 29:313–326.

12. Bartoli, C., M. Civatte, J. F. Pellissier, and D. Figarella-Branger. 2001. CCR2Aand CCR2B, the two isoforms of the monocyte chemoattractant protein-1 re-ceptor are up-regulated and expressed by different cell subsets in idiopathicinflammatory myopathies. Acta Neuropathol. 102: 385–392.

13. Lu, H., D. Huang, N. Saederup, I. F. Charo, R. M. Ransohoff, and L. Zhou. 2011.Macrophages recruited via CCR2 produce insulin-like growth factor-1 to repairacute skeletal muscle injury. FASEB J. 25: 358–369.

14. Lu, H., D. Huang, R. M. Ransohoff, and L. Zhou. 2011. Acute skeletal muscleinjury: CCL2 expression by both monocytes and injured muscle is required forrepair. FASEB J. 25: 3344–3355.

15. Zhang, L., L. Ran, G. E. Garcia, X. H. Wang, S. Han, J. Du, and W. E. Mitch.2009. Chemokine CXCL16 regulates neutrophil and macrophage infiltration intoinjured muscle, promoting muscle regeneration. Am. J. Pathol. 175: 2518–2527.

16. Qian, B. Z., and J. W. Pollard. 2010. Macrophage diversity enhances tumorprogression and metastasis. Cell 141: 39–51.

17. Chawla, A. 2010. Control of macrophage activation and function by PPARs.Circ. Res. 106: 1559–1569.

18. Lawrence, T., and G. Natoli. 2011. Transcriptional regulation of macrophagepolarization: enabling diversity with identity. Nat. Rev. Immunol. 11: 750–761.

19. Farini, A., C. Sitzia, C. Navarro, G. D’Antona, M. Belicchi, D. Parolini, G. DelFraro, P. Razini, R. Bottinelli, M. Meregalli, and Y. Torrente. 2012. Absence of Tand B lymphocytes modulates dystrophic features in dysferlin deficient animalmodel. Exp. Cell Res. 318: 1160–1174.

20. Wehling-Henricks, M., S. Sokolow, J. J. Lee, K. H. Myung, S. A. Villalta, andJ. G. Tidball. 2008. Major basic protein-1 promotes fibrosis of dystrophic muscleand attenuates the cellular immune response in muscular dystrophy. Hum. Mol.Genet. 17: 2280–2292.

21. Kohno, S., T. Ueji, T. Abe, R. Nakao, K. Hirasaka, M. Oarada, A. Harada-Sukeno, A. Ohno, A. Higashibata, R. Mukai, et al. 2011. Rantes secreted frommacrophages disturbs skeletal muscle regeneration after cardiotoxin injection inCbl-b-deficient mice. Muscle Nerve 43: 223–229.

22. Zhang, L., X. H. Wang, H. Wang, J. Du, and W. E. Mitch. 2010. Satellite celldysfunction and impaired IGF-1 signaling cause CKD-induced muscle atrophy.J. Am. Soc. Nephrol. 21: 419–427.

23. Joe, A. W., L. Yi, A. Natarajan, F. Le Grand, L. So, J. Wang, M. A. Rudnicki, andF. M. Rossi. 2010. Muscle injury activates resident fibro/adipogenic progenitorsthat facilitate myogenesis. Nat. Cell Biol. 12: 153–163.

24. Jost, M. M., E. Ninci, B. Meder, C. Kempf, N. Van Royen, J. Hua, B. Berger,I. Hoefer, M. Modolell, and I. Buschmann. 2003. Divergent effects of GM-CSFand TGFbeta1 on bone marrow-derived macrophage arginase-1 activity, MCP-1expression, and matrix metalloproteinase-12: a potential role during arterio-genesis. FASEB J. 17: 2281–2283.

25. Fung-Leung, W. P., M. W. Schilham, A. Rahemtulla, T. M. K€undig,M. Vollenweider, J. Potter, W. van Ewijk, and T. W. Mak. 1991. CD8 is neededfor development of cytotoxic T cells but not helper T cells. Cell 65: 443–449.

26. Kohno, S., Y. Yamashita, T. Abe, K. Hirasaka, M. Oarada, A. Ohno, S. Teshima-Kondo, A. Higashibata, I. Choi, E. M. Mills, et al. 2012. Unloading stressdisturbs muscle regeneration through perturbed recruitment and function ofmacrophages. J. Appl. Physiol. 112: 1773–1782.

27. Segawa, M., S. Fukada, Y. Yamamoto, H. Yahagi, M. Kanematsu, M. Sato, T. Ito,A. Uezumi, S. Hayashi, Y. Miyagoe-Suzuki, et al. 2008. Suppression of mac-

The Journal of Immunology 5159

by guest on June 28, 2018http://w

ww

.jimm

unol.org/D

ownloaded from

rophage functions impairs skeletal muscle regeneration with severe fibrosis. Exp.Cell Res. 314: 3232–3244.

28. Chazaud, B., M. Brigitte, H. Yacoub-Youssef, L. Arnold, R. Gherardi, C. Sonnet,P. Lafuste, and F. Chretien. 2009. Dual and beneficial roles of macrophagesduring skeletal muscle regeneration. Exerc. Sport Sci. Rev. 37: 18–22.

29. Summan, M., G. L. Warren, R. R. Mercer, R. Chapman, T. Hulderman, N. VanRooijen, and P. P. Simeonova. 2006. Macrophages and skeletal muscle regen-eration: a clodronate-containing liposome depletion study. Am. J. Physiol. Regul.Integr. Comp. Physiol. 290: R1488–R1495.

30. Gordon, S. 2003. Alternative activation of macrophages. Nat. Rev. Immunol. 3:23–35.

31. Weisser, S. B., K. W. McLarren, E. Kuroda, and L. M. Sly. 2013. Generation andcharacterization of murine alternatively activated macrophages. Methods Mol.Biol. 946: 225–239.

32. Gordon, S., and P. R. Taylor. 2005. Monocyte and macrophage heterogeneity.Nat. Rev. Immunol. 5: 953–964.

33. Ferrari, G., G. Cusella-De Angelis, M. Coletta, E. Paolucci, A. Stornaiuolo,G. Cossu, and F. Mavilio. 1998. Muscle regeneration by bone marrow-derivedmyogenic progenitors. Science 279: 1528–1530.

34. Uezumi, A., K. Ojima, S. Fukada, M. Ikemoto, S. Masuda, Y. Miyagoe-Suzuki,and S. Takeda. 2006. Functional heterogeneity of side population cells in skeletalmuscle. Biochem. Biophys. Res. Commun. 341: 864–873.

35. Torrente, Y., M. Belicchi, M. Sampaolesi, F. Pisati, M. Meregalli, G. D’Antona,R. Tonlorenzi, L. Porretti, M. Gavina, K. Mamchaoui, et al. 2004. Human cir-culating AC133(+) stem cells restore dystrophin expression and amelioratefunction in dystrophic skeletal muscle. J. Clin. Invest. 114: 182–195.

36. Relaix, F., and P. S. Zammit. 2012. Satellite cells are essential for skeletal muscleregeneration: the cell on the edge returns centre stage. Development 139: 2845–2856.

37. Murphy, M. M., J. A. Lawson, S. J. Mathew, D. A. Hutcheson, and G. Kardon.2011. Satellite cells, connective tissue fibroblasts and their interactions arecrucial for muscle regeneration. Development 138: 3625–3637.

38. Kuang, S., S. B. Charge, P. Seale, M. Huh, and M. A. Rudnicki. 2006. Distinct

roles for Pax7 and Pax3 in adult regenerative myogenesis. J. Cell Biol. 172: 103–

113.39. Gnocchi, V. F., R. B. White, Y. Ono, J. A. Ellis, and P. S. Zammit. 2009. Further

characterisation of the molecular signature of quiescent and activated mouse

muscle satellite cells. PLoS ONE 4: e5205.40. Seale, P., L. A. Sabourin, A. Girgis-Gabardo, A. Mansouri, P. Gruss, and

M. A. Rudnicki. 2000. Pax7 is required for the specification of myogenic sat-

ellite cells. Cell 102: 777–786.41. Sambasivan, R., R. Yao, A. Kissenpfennig, L. Van Wittenberghe, A. Paldi,

B. Gayraud-Morel, H. Guenou, B. Malissen, S. Tajbakhsh, and A. Galy. 2011.

Pax7-expressing satellite cells are indispensable for adult skeletal muscle re-

generation. Development 138: 3647–3656.42. Chazaud, B., C. Sonnet, P. Lafuste, G. Bassez, A. C. Rimaniol, F. Poron,

F. J. Authier, P. A. Dreyfus, and R. K. Gherardi. 2003. Satellite cells attract

monocytes and use macrophages as a support to escape apoptosis and enhance

muscle growth. J. Cell Biol. 163: 1133–1143.43. Warren, G. L., L. O’Farrell, M. Summan, T. Hulderman, D. Mishra, M. I. Luster,

W. A. Kuziel, and P. P. Simeonova. 2004. Role of CC chemokines in skeletal

muscle functional restoration after injury. Am. J. Physiol. Cell Physiol. 286:

C1031–C1036.44. Martinez, C. O., M. J. McHale, J. T. Wells, O. Ochoa, J. E. Michalek,

L. M. McManus, and P. K. Shireman. 2010. Regulation of skeletal muscle re-

generation by CCR2-activating chemokines is directly related to macrophage

recruitment. Am. J. Physiol. Regul. Integr. Comp. Physiol. 299: R832–R842.45. Sun, D., C. O. Martinez, O. Ochoa, L. Ruiz-Willhite, J. R. Bonilla,

V. E. Centonze, L. L. Waite, J. E. Michalek, L. M. McManus, and P. K. Shireman.

2009. Bone marrow-derived cell regulation of skeletal muscle regeneration.

FASEB J. 23: 382–395.

5160 CD8 T CELLS REGULATE SKELETAL MUSCLE REGENERATION

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.jimm

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