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Mammalian skeletal muscle is capable of repairing itself following exercise or injury. This remarkable regenerative capacity relies on satellite cells.1, 2, 3, 4, 5 Normally, satellite cells are kept underneath the basal lamina in a quiescent state. Upon muscle damage or disease, these quiescent stem cells immediately become activated, proliferate, migrate to the injured site, and differentiate to fuse with damaged myofibers or to form new myofibers.1, 2, 3, 4 The regeneration of adult skeletal muscle is a highly coordinated process involving a variety of cell types and signaling molecules that work systematically to repair the damaged myofibers.2, 6, 7, 8 However, how this process is regulated by muscle stem cell niche cues, such as inflammatory signals after muscle injury, still remains elusive.

Many stages of adult muscle regeneration are very similar to embryonic muscle development.1, 9, 10, 11 However, during adult muscle regeneration after acute injury, extrinsic factors are markedly different from those during embryonic development. The most notable and probably the most significant source of such extrinsic factors is the large number of inflammatory cells that infiltrate shortly after muscle damage.8, 12, 13, 14, 15, 16 It has been known that various inflammatory cells can profoundly affect the activation, migration, and differentiation of satellite cells, but the critical roles of inflammatory cells in maintaining skeletal muscle homeostasis have only recently begun to be appreciated.8, 14, 16, 17 Myeloid lineage cells, such as macrophages and the monocytes from which they are derived, are the major inflammatory cells recruited into injured skeletal muscle, and they are unique effector cells in innate immunity.15, 16 Following an early transient recruitment of neutrophils and mononuclear cells derived from circulating monocytes, these macrophages are primed by the inflammatory milieu, which includes local growth factors and cytokines, and begin to polarize into pro-inflammatory classically activated (M1-type) or anti-inflammatory alternatively activated (M2-type) macrophages, which differ in their markers, functions, and cytokine expression profiles.8, 14, 15, 16, 18 Normally, M1 macrophages first accumulate in the injured muscle tissues and produce high levels of inflammatory cytokines, which aid the clearance of apoptotic or necrotic cells and debris. The subsequent transition of myeloid infiltration into anti-inflammatory M2 macrophages is critical for the overall resolution of inflammation in the injured muscles.8, 14, 15, 16, 18 Therefore, loss of balance between these two different types of macrophages would severely compromise healing and regeneration of injured muscle.

miRNAs are small non-coding RNAs that are evolutionarily conserved from plants to mammals.19 Changes in miRNA expression have been associated with various muscle-wasting diseases, such as muscular dystrophies, and several miRNAs have been shown to exacerbate or prevent muscle disease progression in various mouse models of muscular dystrophies, and affect muscle regeneration.20, 21, 22, 23, 24, 25, 26, 27, 28 Furthermore, gain- and loss-of-function studies of miRNAs have clearly demonstrated their important roles in skeletal muscle regeneration and various muscle disorders.20, 26, 27, 29, 30 However, whether a miRNA can affect muscle regeneration by modulating myeloid cells in injured muscle is not well studied.

We have previously reported that microRNA-155 (miR-155) represses myogenic differentiation by targeting MEF2A, a key myogenic transcription factor, in C2C12 cells.31 Processed from the B-cell integration cluster gene (now designated the MIR-155 host gene or MIR-155HG), miR-155 is one of the best-characterized miRNAs, and numerous reports have indicated that miR-155 has a pivotal role in the immune system, particularly in hematopoietic cells upon virus or bacterial infection.32, 33, 34, 35, 36, 37, 42, 78 All experiments with mice were performed according to protocols approved by the Institutional Animal Care and Use Committees of Boston Children's Hospital.

Cardiotoxin injury

Cardiotoxin from Naja Mossambica mossambica (Sigma-Aldrich, St. Louis, MO, USA) was dissolved in sterile saline to a final concentration of 10 μM. In total, 50 μl of cardiotoxin were injected with a 27 Gauge needle into one TA muscle; the other muscle was injected with saline as control.

miRNA mimic in vivo transfection

Injection of miR-155 mimics into the TA muscles of young adult mice was adapted from previous reports.29, 61 In total, 50 μl of microRNA complex was injected into the TA muscle 12 h after cardiotoxin injection and mice were analyzed at the indicated time points.

Histological analysis of skeletal muscles

Skeletal muscles were dissected out and fixed in 4% paraformaldehyde and processed for Hematoxylin and Eosin, Sirius Red, and Fast Green staining as previously described.79 For immunofluorescent staining, skeletal muscle groups were harvested and freshly frozen in liquid nitrogen cooled isopentane (Sigma-Aldrich) and then cryo-embedded in Tissue-Tek OCT medium (Sakura Finetek Inc., Torrance, CA, USA). Muscles were sectioned on a cryostat at 10 μm thickness and placed on permafrost slides (VWR Scientific, Radnor, PA, USA). Images were taken with a Zeiss SteREO Discovery V8 stereomicroscope.

Immunohistochemistry and immunofluorescence

Frozen muscle sections were fixed in 4% paraformaldehyde and permeabilized in 0.5% Triton X-100 for 10 min. Sections were incubated with mouse IgG-blocking solution from the M.O.M kit (Vector Lab, Burlingame, CA, USA) according to the manufacturer's protocol. Primary and secondary antibodies were as following: Desmin (1 : 200, Santa Cruz, Dallas, TX, USA), dystrophin (1 : 200, Sigma-Aldrich), Laminin (1 : 500, Sigma-Aldrich), MF20 (1 : 10, DSHB), Pax7 (1 : 100, DSHB), eMHC (1 : 200, DSHB), BA-F18 (1 : 2, DSHB), BAD5 (1 : 2, DSHB), and Rabbit anti β-galactosidase (1 : 500, Sigma-Aldrich). FITC-conjugated F4/80 and CD11b (eBiosciences, San Diego, CA, USA) were used for staining macrophage markers in cardiotoxin injured TA muscles. All secondary antibodies were obtained from Invitrogen (Carlsbad, CA, USA) and used at 1 : 500 dilutions. Pictures were taken with a Nikon TE2000 epifluorescent microscope with deconvolution (Volocity; Perkin-Elmer, Waltham, MA, USA) or an Olympus FV1000 confocal microscopy (FV1000, Olympus, Center Valley, PA, USA).

Primary myoblast isolation, culture, and differentiation

Primary myoblasts were isolated from neonatal mice as previously described.47 Primary myoblasts were further enriched by pre-plating 30 min for each passage until ~100% cells were positive for desmin. Primary myoblasts were kept in Ham’s F-10 nutrient mixture based growth medium containing 20% fetal bovine serum (FBS), 2.5% chicken embryo extract (USbiologicals, Salem, MA, USA), 5 ng/ml bFGF (Promega, Madison, WI, USA), 100 U/ml penicillin, and 100 μg/ml streptomycin. Differentiation medium (Dulbecco’s Modified Eagle’s Medium; DMEM containing 2% horse serum) was used to induce primary myoblast differentiation.

Macrophage proliferation, viability, and migration assays

Murine macrophage cell line RAW 264.7 was purchased from American Type Culture Collection. Cells were maintained in DMEM (high and low glucose, respectively) supplemented with 10% heat-inactivated FBS (Life Technologies, Carlsbad, CA, USA), 2 mM l-glutamine, 100 U/ml penicillin, and 100 μg/ml streptomycin in a humidified incubator at 37 °C under 5% CO2. Proliferation of RAW 264.7 cells was measured using Click-iT cell proliferation assay kit (Invitrogen) according to manufacturer’s instructions. A final concentration of 30 nM microRNA LNA (locked nucleic acid) inhibitor of miR-155 and negative control oligonucleotide (Dharmacon, Lafayette, CO, USA) were transfected into RAW 264.7 cells using Lipofectamine RNAiMAX (Invitrogen) transfection reagent. After 6 h transfection, the cultures were changed to fresh medium. EdU (5-ethynyl-2′-deoxyuridine, Invitrogen) was added, and 30 h later cells were fixed and harvested for immunohistochemistry analyses. Viability of the cells were assayed using Invitrogen Countess automatic cell counter 72 h after miR-155 or control LNA transfection, with trypan blue dye as indicator of live or dead cells.

Migration assays were performed in Transwell plates (Corning Costar, Tewksbury, MA, USA) with a 6.5-mm diameter and an 8-μm pore size for the membrane.

The RAW 264.7 cells were seeded in six-well plates and transfected with control and miR-155 LNA for 48 h before being transferred into Transwell plates. Fresh medium was added to the bottom well with or without LPS immediately before adding 1 × 105 RAW 264.7 cells into the top well. Plates were incubated for an additional 12 h at 37 °C in a humidified 5% CO2 incubator. Cells in the top wells were wiped off with cotton tips, fixed using 3.7% formaldehyde for 5 min, and stained with DAPI for identifying cell nuclei. The number of cells migrating to the lower membrane surface was then counted, and these data were used for further two-tailed student’s t-test analysis.

Single myofiber isolation

Single myofibers were isolated from the EDL muscle as previously described.80 For immediate quantification of satellite cells, single fibers were fixed in 4% paraformaldehyde for 10 min at room temperature. Fibers were permeabilized with PBST (PBS with 0.5% Triton X-100) for 15 min and blocked with blocking solution (2% BSA/5% goat serum/0.1% Triton X-100 in PBS) for 1 h at room temperature.

FACS and MACS analysis

At indicated time points after cardiotoxin injury, TA muscles were dissected, minced, and digested with STEMxyme2 (Worthington Biochemical Corp., Lakewood, NJ, USA). Cell suspension was then serially filtered through 70 and 40 μm nylon meshes (BD Falcon, Franklin Lakes, NJ, USA). All FACS analyses were performed at the Dana-Farber Cancer Institute flow cytometry facilities with a BD FACSAria II SORP UV sorter. Flowjo software was used to analyze the FACS data. Antibodies used include Pacific Blue conjugated anti-mouse CD45 (Biolegend, 30F-11), Alexa 647 conjugated anti-mouse CD68 (Biolegend, San Diego, CA, USA, FA-11), and Brilliant Violet 605 anti-mouse CD206 Antibody (Biolegend, C068C2). For MACS, cells were incubated with FITC-labeled F4/80 and CD11b antibody (eBiosciences) for 30 min in 4 °C and magnetically separated using an EasySep Mouse FITC Positive Selection Kit (Stemcell Technology, Vancouver, BC, Canada). Cell numbers were determined using Countess automated cell counter (Invitrogen).

RT-PCR, real-time PCR, and taqman assays

miRNAs and total RNAs were extracted using Trizol and were cleaned using miRNeasy kit (Qiagen, Valencia, CA, USA). miRNAs were measured using Taqman MicroRNA Reverse Transcription Kit and Taqman Universal Master Mix Kit (Applied Biosystems, Carlsbad, CA, USA). All SYBR-based real-time PCRs were run on a CFX96 or CFX384 Real-Time PCR machine (Bio-Rad, Hercules, CA, USA) with iScript reverse transcription kit and iTaq supermix (Bio-Rad). A list of SYBR-based real-time PCR primers can be found in the Supplementary Table.

Western blot analysis

Total muscle protein was extracted using RIPA buffer containing protease inhibitor cocktail (Roche, Indianapolis, IN, USA) and 1 mM PMSF. Protein concentrations were measured by a DC protein assay (Bio-Rad). Western blotting was performed by standard protocol. The following antibodies were used: SOCS1 (1 : 1000, Cell signaling, Danvers, MA, USA), phospho-STAT3 (1 : 1000, Cell signaling), anti-mouse STAT3 (1 : 1000, Cell signaling), γ-tubulin (1 : 5000, Sigma-Aldrich), GAPDH (1 : 5000, Sigma-Aldrich). Primary antibody was visualized with either IRDye 680RD goat anti-mouse or IRDye 800CW goat anti-rabbit (LI-COR, Lincoln, NE, USA) on the Odyssey imaging system (LI-COR Biosciences).

Plasmids, transfection and luciferase assays

The 3'-UTR fragment of mouse SOCS1 containing miR-155 binding sites was cloned into the pMIR-glow vector (Promega, Madison, WI, USA). miR-155 sensor and mutagenesis of the miR-155 binding sites were performed as previously described.42 Hek293T cells (CRL-11268;ATCC) were grown in DMEM containing 10% FBS. Transfection was performed with Lipfectamine 2000 reagents (Invitrogen) according to the manufacturer's instructions. For luciferase assays, Firefly and Renila luciferase activity were measured using Dual-Glo Luciferase Assay kit (Promega) according to the manufacturer's instructions. All experiments were performed in triplicate and were repeated at least twice.

Statistics

Unless otherwise stated, all statistical analyses were performed using unpaired two-tailed student's t-test. Data are presented as mean value or percentage change±S.E.M. P<0.05 was considered to be statistical significance.