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SHP-1−Dependent Macrophage Differentiation Exacerbates -Induced Myositis

This information is current as Neva B. Watson, Karin M. Schneider and Paul T. Massa of September 26, 2021. J Immunol 2015; 194:2796-2809; Prepublished online 13 February 2015; doi: 10.4049/jimmunol.1402210 http://www.jimmunol.org/content/194/6/2796 Downloaded from

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The Journal of Immunology is published twice each month by The American Association of Immunologists, Inc., 1451 Rockville Pike, Suite 650, Rockville, MD 20852 Copyright © 2015 by The American Association of Immunologists, Inc. All rights reserved. Print ISSN: 0022-1767 Online ISSN: 1550-6606. The Journal of Immunology

SHP-1–Dependent Macrophage Differentiation Exacerbates Virus-Induced Myositis

Neva B. Watson,* Karin M. Schneider,* and Paul T. Massa*,†

Virus-induced myositis is an emerging global affliction that remains poorly characterized with few treatment options. Moreover, muscle-tropic often spread to the CNS, causing dramatically increased morbidity. Therefore, there is an urgent need to explore genetic factors involved in this class of human disease. This report investigates critical innate immune pathways affecting murine virus–induced myositis. Of particular importance, the key immune regulator src homology region 2 domain–containing phosphatase 1 (SHP-1), which normally suppresses macrophage-mediated inflammation, is a major factor in promoting clinical disease in muscle. We show that Theiler’s murine encephalomyelitis virus (TMEV) infection of skeletal myofibers induces inflammation and subsequent dystrophic calcification, with loss of ambulation in wild-type (WT) mice. Surprisingly, although 2/2 similar extensive myofiber infection and inflammation are observed in SHP-1 mice, these mice neither accumulate dead Downloaded from calcified myofibers nor lose ambulation. Macrophages were the predominant effector cells infiltrating WT and SHP-12/2 muscle, and an increased infiltration of immature monocytes/macrophages correlated with an absence of clinical disease in SHP-12/2 mice, whereas mature M1-like macrophages corresponded with increased myofiber degeneration in WT mice. Furthermore, blocking SHP-1 activation in WT macrophages blocked virus-induced myofiber degeneration, and pharmacologic ablation of macrophages inhibited muscle calcification in TMEV-infected WT animals. These data suggest that, following TMEV infection of

muscle, SHP-1 promotes M1 differentiation of infiltrating macrophages, and these inflammatory macrophages are likely involved http://www.jimmunol.org/ in damaging muscle fibers. These findings reveal a pathological role for SHP-1 in promoting inflammatory macrophage differ- entiation and myofiber damage in virus-infected skeletal muscle, thus identifying SHP-1 and M1 macrophages as essential mediators of virus-induced myopathy. The Journal of Immunology, 2015, 194: 2796–2809.

revalent human ssRNA viruses, including , were described as targets for initial replication in the periphery flaviviruses, , , and rhabdovi- (22–29). Moreover, recent studies suggested that disease severity P ruses, are commonly associated with skeletal muscle in- of Chikungunya virus strains can be predicted by the ability of fection and inflammation resulting from either direct infection of each strain to infect myofibers (30, 31). Therefore, it is common myofibers or infiltrating inflammatory macrophages (1–15). As that muscle serves as an initial target for neurotropic viruses. by guest on September 26, 2021 a consequence of these infections, spreading of the virus from Another important aspect of these virus-induced neuromuscular muscle to CNS by retrograde axonal transport or viremia may diseases is the particular targeting of children. Many childhood occur, causing severe neuropathological complications (16–20). viral infections trigger CNS disease and/or muscle dysfunction, Therefore, skeletal muscle is a clinically significant target of virus with severe cases of muscle involvement manifesting in severe infections in humans, and the mechanisms involved in this process inflammatory myositis and/or rhabdomyolysis (32–35). Thus, virus must be elucidated further. infections involving skeletal muscle with common subsequent Among the most clinically relevant muscle-tropic viruses, neurologic involvement are an escalating clinical problem with arthropod-borne RNA viruses (arboviruses), including flaviviruses increased predilection for children. Therefore, defined animal and alphaviruses, are emerging disease threats worldwide in both models for these infections are needed to study genetic and im- developing and developed nations, and these infections are asso- mune mechanisms at play in the development of severe virus- ciated with a high incidence of neurologic manifestations (21). induced muscle and subsequent CNS disease. Alphaviruses, such as Ross River virus, Chikungunya virus, and Theiler’s murine encephalomyelitis virus (TMEV) is a neuro- Sindbis virus, were demonstrated to trigger debilitating muscle tropic belonging to the cardiovirus genus that is and CNS disease in humans and mice, and skeletal muscle fibers made up of two subgroups (36). GDVII, the highly virulent strain, causes an acute paralytic disease with infection of neurons in the brain and spinal cord leading to and death. Addi- *Department of Microbiology and Immunology, State University of New York Upstate Medical University, Syracuse, NY 13210; and †Department of Neurology, tionally, GDVII was reported to induce myositis marked by ex- State University of New York Upstate Medical University, Syracuse, NY 13210 tensive myofiber necrosis following i.m. inoculation (36–38). In Received for publication August 29, 2014. Accepted for publication January 8, 2015. contrast, attenuated strains of TMEV, including BeAn and DA, are This work was supported by National Institutes of Health Grant R01 NS072051. only neurotropic when inoculated intracranially into susceptible Address correspondence and reprint requests to Dr. Paul T. Massa, State University of strains of adult mice (36). These attenuated strains of TMEV are New York Upstate Medical University, 750 East Adams Street, WH 2237B, Syracuse, commonly used as a virus-induced model of multiple sclerosis, NY 13210. E-mail address: [email protected] often causing subclinical neuronal infection followed by persis- Abbreviations used in this article: BL/6, C57BL/6J; C3H, C3HeB/FeJLe; me/me, motheaten; mev/mev, viable motheaten; MFI, mean fluorescence intensity; P.I., post- tence in glia and inflammatory demyelination weeks to months infection; SAPE, streptavidin-PE; SHP-1, src homology region 2 domain–containing postinfection (P.I.) (36). Importantly, attenuated TMEV strains phosphatase 1; TMEV, Theiler’s murine encephalomyelitis virus; WT, wild-type. were demonstrated to induce a severe acute myositis following Copyright Ó 2015 by The American Association of Immunologists, Inc. 0022-1767/15/$25.00 i.m. inoculation and to induce an immune cell–mediated acute www.jimmunol.org/cgi/doi/10.4049/jimmunol.1402210 The Journal of Immunology 2797 myositis following i.p. inoculation into several strains of suckling No. CCL-10). Plaque assays were performed to determine viral titer as PFU/ mice (38, 39). These studies indicate that TMEV infection of ml. Twelve-day-old me/me mice and normal littermates were inoculated i.p. 3 6 suckling mice may provide a model system for studying the with 100 ml containing 6 10 PFU BeAn TMEV. Mice were monitored daily for signs of skeletal muscle disease (hunched, abnormal gait, swollen pathogenesis of virus-induced muscle disease that may be relevant shoulders/hips). WT mice (n = 16) were sacrificed upon clinical signs of for increased childhood susceptibility to these viruses. muscle disease (9–15 d P.I.) unless otherwise noted, and development of We previously established that suckling Src homology region 2 skeletal muscle disease was verified by the presence of white calcified domain–containing phosphatase 1 (SHP-1)–deficient mice (moth- areas within skeletal muscle upon dissection. Alternatively, WT and me/me littermates were sacrificed at 5 d P.I. and processed for RNA, histological, eaten [me/me]) suffer CNS disease following intracranial or pe- and flow cytometry analysis. ripheral infection with the attenuated BeAn strain of TMEV, Twelve- to fourteen-day-old mev/mev mice and normal littermates were whereas wild-type (WT) mice are resistant to this disease (40– inoculated i.p. with 100 ml containing 6 3 105 to 3 3 106 PFU TMEV. Infections were independently repeated five times: once with 3 mev/mev 42). In this model, macrophages were found to mediate TMEV- v v induced demyelination in SHP-12/2 mice, because blocking and 3 WT animals; once with 1 me /me and 2 WT animals; twice with 1mev/mev and 5 WT animals; and once with 1 mev/mev and 1 WT animal, macrophage infiltration inhibited TMEV infection of the CNS, yielding 7 total mev/mev mice and 16 total WT littermates. Mice were corresponding with reduced disease severity (40, 41). SHP-1 is monitored daily for signs of skeletal muscle disease (hunched, abnormal a cytosolic protein tyrosine phosphatase expressed predominantly gait, swollen shoulders/hips). Mice were sacrificed upon clinical signs of in hematopoietic cells, and it functions as a key regulator of many muscle disease (7–15 d P.I.) unless otherwise noted. Development of skeletal muscle disease was verified by the presence of white calcified cellular processes of innate immune cells, especially in macro- areas within skeletal muscle upon dissection. phages (43, 44). In particular, SHP-1 controls essential monocyte/ macrophage functions, including transendothelial migration, adhe- Histology Downloaded from sion, activation, polarization, and phagocytic activity, which are rele- Skeletal muscle was dissected from the gluteus maximus muscle, snap vant to virus-induced inflammatory responses in tissues (44–46). frozen in OCT media (Sakura Finetek USA, Torrance, CA), and stored at We report that both WT and me/me suckling mice develop 280˚C. Seven-micron sections were cut using a cryostat (Leica widespread myofiber infection and innate inflammatory responses Microsystems CM1900, Wetzlar, Germany), air-dried, and fixed with acetone (10 min at 220˚C). in skeletal muscles upon peripheral inoculation with TMEV. Sections for immunofluorescent staining were blocked in 10% horse

However, TMEV only induces severe myositis with impaired lo- serum (Tissue Culture Biologicals, Tulare, CA; No. 601D) overnight at 4˚C http://www.jimmunol.org/ comotion following peripheral inoculation of WT mice; mice and incubated in 10% horse serum with primary Abs overnight at 4˚C. Rat lacking SHP-1 are surprisingly resistant to this clinical muscle anti-mouse Ly6c and F4/80 (BioLegend, San Diego, CA; No. 128001 and pathology. In particular, an accumulation of degenerating fibers and 123101) were diluted at 1:200, and rabbit antiserum against BeAn TMEV (a gift from Howard L. Lipton, University of Illinois at Chicago, Chicago, corresponding dystrophic fiber calcification were observed in WT, IL) was diluted at 1:2000. Fluorochrome-conjugated secondary Abs but not SHP-1–deficient, mice. Importantly, although TMEV in- (Dylight 488 donkey anti-rabbit and Dylight 594 goat anti-rat) were ob- fection of WT and me/me muscle induces a pronounced infiltra- tained from Jackson ImmunoResearch Laboratories (West Grove, PA; No. tion of macrophages, there are clear differences in the phenotype 711-545-152 and 112-515-167, respectively) and incubated for 2 h at 4˚C at a 1:500 dilution. DAPI (1 mg/ml) was added for 1 min, and coverslips of these cells between WT and me/me mice that relate to striking were mounted with fluorescence mounting medium (DAKO, Glostrup, differences in clinical muscle pathology. Of particular note, Denmark; No. S3023). by guest on September 26, 2021 muscle pathology in WT animals was coincident with macrophage H&E staining was performed with all steps at room temperature, unless differentiation toward an M1 phenotype, whereas muscle- otherwise noted. Tissues were incubated with Harris Hematoxylin (Sigma infiltrating macrophages displayed a relatively immature pheno- Diagnostics, St. Louis, MO; No. HHS-32) for 11 min in a slide warmer (Lab-Line Instruments, Melrose Park, IL; No. 26025) at 50˚C. Slides were type in me/me mice. Additionally, TMEV genomes were much decolorized with 0.3% HCl in 70% EtOH for ∼50 s, washed in ammonium higher in WT macrophages compared with me/me macrophages, water (five drops ammonium hydroxide/100 ml dH2O) for 1 min, and suggesting that an M1-differentiation program supported virus stained with Eosin Y (Sigma Diagnostics; No. HT110-1-16) for 45 s. replication in these cells within muscle. Furthermore, blocking Slides were dried in a 50˚C slide warmer for 5 min and mounted using Permount (Electron Microscopy Sciences, Hatfield, PA; No. 17986-01). SHP-1 activation in WT mice via the CD47–SIRPa–SHP-1 Alizarin red S (Electron Microscopy Sciences; No. 26206-01) staining pathway blocked the development of both myofiber calcification was performed on acetone-fixed sections, according to the manufacturer’s and impaired ambulation. Finally, depleting macrophages in WT recommended protocol. Tissues were stained with Alizarin Red S for animals with clodronate liposomes inhibited the development of 2.5 min. Slides were dried on a 50˚C slide-warmer and mounted with TMEV-induced muscle fiber degeneration and calcification. Thus, Permount. Picrosirius red (Electron Microscopy Sciences; No. 26357-02) staining these findings demonstrate that SHP-1 plays a critical role in was performed on acetone-fixed sections according to the manufacturer’s promoting M1 differentiation in virus-infected muscle and in protocol. Phosphomolybdic acid was added to tissues for 5 min. Slides promoting debilitating virus-induced myositis. were dehydrated in EtOH, cleared in xylene, and mounted with Permount. Bright-field and fluorescent microscopy were performed using an Eclipse E800 microscope (Nikon, Tokyo, Japan) with a SPOT RT Slider digital Materials and Methods camera (SPOT Imaging Solutions, Sterling Heights, MI). Images were Animals obtained at original magnifications 320, 3200, and 3400, with numerical apertures of 0.06, 0.50, and 0.75, respectively, using Spot RT Software SHP-1–deficient me/me (C3FeLe.B6 a/a-Ptpn6me/J) mice or viable moth- v3.0 (SPOT Imaging Solutions) at room temperature. Black and white eaten (mev/mev; C57BL/6J [BL/6]-Ptpn6me-v/J) mice, as well as hetero- fluorescent images were merged and pseudo-colored using ImageJ soft- zygous and WT littermates, were produced from heterozygous breeding ware (National Institutes of Health, Bethesda, MD). pairs obtained from The Jackson Laboratory (Bar Harbor, ME; No. 000225 H&E cell counts were executed by placement of rectangles of a fixed and 000811, respectively). Appropriate genotypes of all mice were verified area over representative regions of each tissue and counting cells within commercially from tail DNA (Transnetyx, Cordova, TN). All animal each area. Unit area for cell density within lesions was 2.25 nm (30 3 75 mm), experiments were performed under approval from the Institutional Animal and unit area for cells invading myofibers was 450 mm(183 25 mm). Areas Care and Use Committee at State University of New York Upstate Medical were placed over two representative fields/tissue, and averaged counts were University. analyzed for statistical significance. Infections Flow cytometry analysis BeAn TMEV was obtained from the American Type Culture Collection At 5 d P.I., all skeletal muscle, including forearms, neck, back, hindlimbs, (Manassas, VA; No. VR-995) and propagated within BHK-21 cells (ATCC and gluteus maximus muscles, was dissected from WT and me/me mice. 2798 SHP-1 IN VIRUS-INDUCED MYOSITIS

Muscle was dissociated using a gentleMACS Dissociator and Skeletal were processed with these probes, according to the QuantiGene Plex 2.0 Muscle Dissociation Kit (Miltenyi Biotec, Cologne, Germany; No. 130- Assay manual. Briefly, a working bead mix containing lysis mixture, 098-305), according to the manufacturer’s protocol. Cells were passed blocking reagent, capture beads, and probe set was prepared and dispensed through 40-mm filters following RBC lysis. Cells were incubated at 4˚C for into the hybridization plate, and 20 ml total RNA containing 500 ng (from 20 min in 2.4G2 supernatant (2.4G2 hybridoma line, ATCC No. HB197) to muscle samples), 300 ng (from muscle-sorted cells), or 50 ng (from spinal block CD16/32 FcgRs. Cells were stained for 30 min at 4˚C with the cord–sorted cells) was added to each well. Background control wells re- following Abs and concentrations diluted in 2.4G2 supernatant: CD45-PE ceived 20 ml water added to the bead mix. The hybridization plate was (No. 103105; 1:100), CD11b–Pacific Blue (No. 101223; 1:800), Ly6c– sealed and placed into a VorTemp 56 shaking incubator (Fisher) for 22 h at Brilliant Violet 570 (No. 128030; 1:50), F4/80-PerCP/Cy5.5 (No. 123127; 54˚C and 600 rpm. 1:50), or CCR2-allophycocyanin (R&D Systems, Minneapolis, MN; No. After hybridization, the wash solution, preamplifier, amplifier, label FAB5538A; 1:10). Unless otherwise noted, all Abs were from BioLegend. probe, and streptavidin-PE (SAPE) solutions were prepared according to the Flow cytometric analysis was performed using a BD LSR Fortessa manual instructions. The hybridized samples were transferred from the cytometer with FACSDiva software (Becton Dickinson, San Jose, CA) and hybridization plate to the magnetic separation plate. Samples were washed analyzed with FlowJo software (TreeStar, Ashland, OR). Experiments using the Affymetrix Hand-Held Magnetic Plate Washer and incubated were performed twice independently. sequentially for 1 h each (50˚C at 600 rpm) with the premade amplifier FACS was performed using a BD FACSAria III cell sorter with solutions (preamplifier, amplifier, and label probe). The SAPE solution was FACSDiva software (Becton Dickinson) to isolate either muscle- or spinal added to the plate, and samples were incubated in the dark at room tem- cord–infiltrating cells for RNA analysis. Muscle-infiltrating cells were perature for 30 min at 600 rpm. Unbound SAPE was removed using the prepared as described above. To isolate spinal cord–infiltrating cells, whole SAPE Wash Buffer from the QuantiGene Plex Kit. Then, 130 ml SAPE spinal cords were dissected and dissociated using a gentleMACS dis- Wash Buffer was added to each sample, and the plate was shaken at room sociator and Neural Tissue Dissociation Kit, according to the manu- temperature for 3 min at 800 rpm to resuspend the beads. The plate was facturer’s protocol (Miltenyi Biotec; No. 130-092-628). Following RBC read immediately using the Bio-Rad BioPlex 200 instrument with settings lysis, myelin was removed from spinal cord cell suspensions using Myelin of 100 ml volume, 60 s timeout, and 100 bead events/bead region. Downloaded from Removal Beads II (Miltenyi Biotec; No. 130-096-733), according to the Fluorescent readings from blank wells were subtracted from fluorescent manufacturer’s protocol. Muscle- and spinal cord–infiltrating cells were values for each mRNA of interest. Values exceeding background were labeled with CD45-PE, CD11b–Pacific Blue, Ly6c–Brilliant Violet 570, normalized to the geometric mean signal derived from three reference genes and F4/80-PerCP/Cy5.5. CD45+ cells were gated and collected for spinal in each sample: GAPDH, HPRT1, and TBP1. These normalized ratios were cords, and muscle cells were subsequently subjected to a generous CD11b+ scaled to positive integer values by multiplying them by a constant (10,000). Ly6c+(hi and lo) gate to yield CD45+CD11b+Ly6c+ cells. A total of 250,000– Significant differences between sample groups were evaluated using a one- 400,000 cells was collected per muscle sample, and 50,000–150,000 cells tailed unpaired t test. were collected per spinal cord. Cells were pelleted, suspended in RNA http://www.jimmunol.org/ STAT-60 (Tel-Test, Friendswood, TX; No. Cs-111), and stored at 280˚C Statistical analyses until further processing for RNA extraction/analysis. GraphPad Prism 5 software was used to perform statistical analyses. One- In vivo anti-CD47 treatment tailed unpaired t tests were performed to compare one measurement be- tween two groups, two-way ANOVAs with Bonferroni post hoc tests for Twelve WT C57BL/6 mice (The Jackson Laboratory; No. 00064) were multiple comparisons were performed to measure significant differences inoculated i.p. with 6 3 106 PFU TMEV at 12 d. Mice were injected i.p. between means 6 SEM, and one-tailed Wilcoxon matched-pairs signed- with 100 mg rat IgG2a, k isotype control or rat anti-CD47 Abs (Bio- rank tests were performed to compare clinical disease scores between two Legend; No. 400516 and 127512, respectively) every 2 d from 5 to 13 d groups. GraphPad Prism 5 software also was used to calculate statistical P.I. and sacrificed at 16 d P.I. Four animals were used for each control and significance of disease incidence using the two-sided Fisher exact test. by guest on September 26, 2021 experimental group. Mice were observed daily for signs of disease, and clinical disease scores were assigned according to the following rank scale: 1, hunched posture and ruffled fur; 2, swollen muscles/abnormal gait; 3, Results one limb immobilized; 4, two or more limbs immobilized; and 5, death. TMEV-induced skeletal muscle disease in WT mice Dissections and histological evaluations were performed as described above. This experiment was performed in duplicate. We reported previously that suckling mice lacking SHP-1 (me/me) The percentages of calcified areas in Alizarin red–stained muscle sec- on a C3H background develop rapid CNS inflammatory disease tions were quantified using ImageJ software. Bright-field images were following i.p. inoculation of the attenuated BeAn TMEV strain converted to 8-bit files. Automatic threshold values were used to select and within 5–8 d P.I (40). Although WT animals remain free of CNS measure areas of calcified (pixelated) staining, threshold values were in- disease, we observed that i.p. inoculation of TMEV to suckling creased to select and measure entire tissue area, and the percentage of tissue calcification was calculated using these area measurements. heterozygous and homozygous WT littermates produced a severe debilitating disease of the skeletal muscle between 9 and 15 d P.I. In vivo macrophage depletion (Fig. 1, Table I). Because no phenotypic difference between het- Ten WT C3HeB/FeJLe (C3H) mice were inoculated i.p. with 6 3 107 PFU erozygous and homozygous WT littermates was seen following TMEV at 12 d. Mice were injected i.p. with 80 ml containing 0.4 mg infection, we refer to both +/2 and +/+ animals as WT in this clodronate or control liposomes (Clodrosome Macrophage Depletion Kit; article. Mice exhibiting TMEV-induced muscle disease suffered Encapsula Nano Sciences, Brentwood, TN; No. 8901) every 3 d from 6 to loss of limb function that was musculoskeletal rather than neu- 12 d P.I. and were sacrificed at 14 d P.I. Five animals received clodronate liposomes, and five animals received control liposomes. Mice were ob- rologic in nature, because clinical examination and dissection served daily for signs of disease, and clinical disease scores were assigned revealed exceptionally stiff, swollen skeletal muscle presenting according to the scale described above. Mice were observed for signs of a white appearance, with limbs completely immobilized in severe muscle calcification upon dissection. cases (Fig. 1). Experiments using WT mice on a BL/6 background demonstrated that TMEV triggered comparable muscle patholo- RNA analysis gies in both C3H and BL/6 mice (Fig. 1), signifying that TMEV- Skeletal muscle was dissected from the gluteus maximus muscle, ho- induced muscle disease was not specific to the C3H strain, in 2 mogenized in RNA STAT-60 (Tel-Test), and stored at 80˚C. FACS-sorted agreement with previous studies (39). Muscle pathology was ab- cells were isolated for RNA analysis, as described above. Samples were processed further for RNA purification by phenol-chloroform extraction, sent in both mock-injected BL/6 and C3H mice (Fig. 1, BL/6 not and RNA were analyzed with a custom-designed QuantiGene Plex 2.0 shown). Assay (Affymetrix, Santa Clara, CA). To determine the pathological basis for this phenotype, H&E The Affymetrix QuantiGene Plex 2.0 Assay (a multiplex bead-based and alizarin red staining were performed on serial skeletal assay) was used to measure the expression of 41 genes of interest (in- cluding 38 target genes and 3 reference genes). Prevalidated probes for these muscle sections from infected mice. The white appearance of genes were ordered directly from Affymetrix and arrived as a conjugated skeletal muscle in WT mice was likely due to calcification, bead mixture. Mouse total RNA samples from muscle or cell-sorted samples because alizarin red staining was strongly positive (Fig. 1). The Journal of Immunology 2799 Downloaded from http://www.jimmunol.org/ by guest on September 26, 2021

FIGURE 1. TMEV-induced muscle pathology in WT mice. Twelve-day-old WT C3H or C57BL/6 mice were inoculated i.p. with 6 3 106 PFU TMEVor mock supernatant (C3H only) and sacrificed at 14–15 d P.I. Representative images of observed skeletal muscle disease upon dissection (top row); red arrows indicate calcified (white) muscle. Serial muscle sections stained with H&E or Alizarin red S (middle two rows); scale bars, 200 mm(n = 4). Muscle sections were stained with picrosirius red (bottom row); scale bars, 50 mm(n = 4).

Moreover, calcification was restricted to degenerating myo- the notion of TMEV-induced dystrophic calcification of skeletal fibers, suggesting that the process of calcification occurred muscle. primarily in fibers undergoing terminal degeneration that eventually led to fiber loss. Accompanying fibrosis was prevalent, Lack of TMEV-induced muscle disease in SHP-1–deficient as observed by picrosirius red staining (Fig. 1), further supporting mice In contrast to WT mice, no overt clinical or anatomical signs of

2/2 TMEV-induced muscle disease were visible in SHP-1–deficient Table I. Frequency of observed muscle disease in WT and SHP-1 me/me mice. This suggested that SHP-1 might facilitate myofiber mice calcification and muscle pathology in WT animals. However, be- cause me/me mice succumb to CNS disease prior to the time at % Mice with Observed Muscle Pathology (Total No. Mice) p Value which muscle disease is clearly discernible in WT mice, we per- formed similar experiments with mev/mev mice (BL/6 back- C3Ha WT (+/+) 80 (5) 1 ground), which develop CNS disease later than do me/me mice WT (+/2) 90.91 (11) (42). As depicted in Fig. 2 and Table I, all BL/6 WT mice BL/6b exhibited signs of muscle calcification by 7 d P.I., whereas cal- WT (+/+; +/2) 100 (16) ,0.0001 v v v v cification was never observed in me /me littermates by the time me /me 0 (7) the mice succumbed to CNS disease (up to 11 d P.I.). Alizarin red aAnimals were infected with 6 3 106 PFU TMEV and sacrificed between 9 and staining confirmed the presence of degenerating calcified myo- 15 d P.I. v v bAnimals were infected with 6 3 105–3 3 106 PFU TMEV and sacrificed be- fibers in WT, but not in me /me , muscle (Fig. 2B), further vali- tween 7 and 11 d P.I. dating sharply reduced myofiber degeneration and muscle 2800 SHP-1 IN VIRUS-INDUCED MYOSITIS

FIGURE 2. Muscle pathology in WT and SHP-12/2 mice. Twelve-day-old mev/mev mice (lower panels) and WT littermates (upper panels) were infected with 6 3 105 PFU TMEV or mock supernatant and sacrificed at 9 d P.I. (A) Representative images of dissected mice; arrows indicate observed muscle disease by apparent calcification. (B) Alizarin red (left panels) or H&E (middle panels) staining was performed on serial skeletal muscle sections; arrows point to corresponding calcified fibers in adjacent sections. Scale bars, 50 mm(n = 3). Muscle sections were stained with picrosirius red (right panels); scale bars, 50 mm(n = 4). Downloaded from pathology in SHP-1–deficient mice. Although mev/mev mice did CD11b+Ly6chi and CD45+CD11b+Ly6clo cells/g of muscle in not show signs of myofiber death/calcification, both mev/mev and SHP-12/2 mice compared with WT mice (Fig. 4B). These data WT littermates exhibited equal signs of muscle fibrosis at 9 d P.I. supported our histological observations, demonstrating a much (Fig. 2B), indicating that myofiber calcification was a specific more robust infiltration of both Ly6chi and Ly6clo monocyte- marker of clinical disease in WT animals. derived cells in SHP-12/2 muscle compared with WT muscle.

To further evaluate potential phenotypic differences between http://www.jimmunol.org/ Phenotypic differences of infiltrating macrophages in WT and 2 2 infiltrating macrophages in WT mice compared with me/me mice, SHP-1 / mice we analyzed levels of the differentiation markers CCR2 and F4/80 A prominent feature of TMEV-infected muscle, both before and on muscle-infiltrating macrophages by flow cytometry. The most during the development of clinical muscle pathology, was the striking differences observed were high levels of CCR2 expression presence of extensive macrophage invasion into infected muscles on CD45+CD11b+Ly6chi cells in me/me mice compared with WT 2/2 of both SHP-1 and WT mice (Fig. 3), whereas increased, yet mice and low levels of F4/80 expression on CD45+CD11b+Ly6clo + relatively low, numbers of Ly6g cells were observed within SHP- macrophages in me/me mice compared with WT mice, as revealed 2/2 1 muscle compared with WT muscle by immunofluorescence by contour plots (Fig. 4C). Consistent with these observations, and flow cytometry analysis (data not shown). Because SHP-1 is CD45+CD11b+Ly6chiF4/80lo cells in me/me mice displayed a sig- by guest on September 26, 2021 expressed at high levels in monocytes/macrophages, we reasoned nificantly increased CCR2 mean fluorescence intensity (MFI), that development of muscle pathology might be controlled by whereas CD45+CD11b+Ly6cloCCR2lo cells in me/me mice dis- 2/2 SHP-1 in these cells. Indeed, in both SHP-1 and WT mice played a significantly decreased F4/80 MFI, compared with the + there was an extensive infiltration of predominantly Ly6c mac- corresponding WT populations (Fig. 4D). These observations rophages that were especially localized to regions of extensive suggested a role for SHP-1 in governing macrophage maturation TMEV-infected myofibers (examples of these regions are depicted following recruitment of Ly6chiCCR2hi inflammatory monocytes in Fig. 3A). This observation suggested that macrophage infiltra- into TMEV-infected muscles, leading to a predominance of rela- 2/2 tion in WT and SHP-1 muscle generally localized to areas of tively immature macrophages in SHP-1–deficient muscle com- TMEV infection of myofibers. Although both genotypes contained pared with WT muscle. abundant infiltrates, macrophage infiltration consistently appeared more robust in SHP-12/2 muscle compared with WT muscle CD47 neutralization attenuates disease severity in WT mice (Fig. 3A). To support this observation, the average densities of SHP-1 controls macrophage activities, including phagocytosis and infiltrating cells accumulating between or within muscle fibers of cytokine production, particularly through the CD47–SIRPa–SHP- infected SHP-12/2 and WT mice were enumerated; cell counts 1 signaling axis following contact with CD47+ targets (46, 51, 52). revealed a higher density of inflammatory cells between myofibers Additionally, CD47–integrin interactions control other important of SHP-12/2 muscle compared with WT muscle (Fig. 3B, 3C). aspects of monocyte functions, including transendothelial migra- To extend the observation that SHP-12/2 mice displayed a more tion, which might explain the increased numbers of macrophages pronounced infiltration of inflammatory monocytes/macrophages in me/me mouse muscle following TMEV infection (52–54). in infected muscle, flow cytometry was performed on cells iso- Therefore, we addressed whether muscle pathology in WT mice lated from WT and me/me muscle at 5 d P.I. It is generally well could be ameliorated by blockade of CD47–SIRPa–SHP-1 sig- understood that CD11b+Ly6chiCCR2hi inflammatory monocytes naling in macrophages. To do this, WT BL/6 mice were injected patrol the bloodstream and respond to damaged/inflamed tissues in with a CD47-neutralizing Ab using an established protocol (55). an MCP-1–dependent manner. Upon entry into inflamed tissues, Because we had observed extensive TMEV infection of skeletal such as skeletal muscle, inflammatory monocytes mature into muscle at 5 d P.I. but had not observed substantial calcification macrophages, first by downregulating CCR2 and subsequently prior to 9 d P.I., we initiated injections of CD47-neutralizing Ab downregulating Ly6c while gaining F4/80 expression (Fig. 4A) 5 d P.I. Mice were treated with anti-CD47 from days 5 to 13 d P.I. (47–50). Therefore, we first enumerated infiltrating CD11b+Ly6chi and were sacrificed at 16 d P.I. No differences in muscle pathology and CD11b+Ly6clo cells within WT and me/me muscle, and CD45 were observed between control groups (untreated or IgG treated); was used as an initial gate to exclude nonhematopoietic cells. We therefore, all control animals were pooled for quantification. Mice observed significantly higher numbers of both infiltrating CD45+ were assigned clinical disease scores daily from 9 to 16 d P.I. As The Journal of Immunology 2801 Downloaded from http://www.jimmunol.org/

FIGURE 3. Infiltration of WT and SHP-12/2 cells into skeletal muscle following infection. (A)mev/mev mice and WT littermates were sacrificed at 9 d by guest on September 26, 2021 P.I., and muscle sections were labeled with Abs against Ly6c (red) and TMEV (green) and stained with DAPI (blue); asterisks denote uninfected myofibers. Image fields within the white boxes in left panels are shown as enlarged images in right panels. Scale bars, 100 mm(n = 3). (B) me/me mice and WT littermates (n = 2) and mev/mev mice and WT littermates (n = 2) were sacrificed at 5 d P.I., and mev/mev mice and WT littermates (n = 4) were sacrificed at 9 d P.I. Cell counts were performed on H&E-stained muscle sections to determine the average number of cells/unit area within lesions (blue rectangles)or within intact myofibers (black rectangles). Representative images of me/me and WT littermates at 5 d P.I. and mev/mev and WT littermates at 9 d P.I. are shown. Scale bars, 50 mm. (C) Graphical representation of the average number of cells/unit area with lesions (upper panel) and average number of cells invading fibers/unit area (lower panel). *p , 0.05, **p , 0.01, ****p , 0.0001. expected, animals receiving anti-CD47 treatments exhibited of SHP-1 in WT macrophages may promote macrophage differ- a significant reduction in clinical disease scores compared with entiation in ways that are detrimental to muscle function. controls (Fig. 5A). Upon dissection, mice receiving CD47- blocking Ab had reduced calcification of skeletal muscle com- Inflammatory M1 macrophages correlate with increased pared with controls (Fig. 5B). Quantification of alizarin red muscle pathology in WT mice staining revealed significantly decreased muscle calcification in Because our data indicated that SHP-1 controlled TMEV-induced anti-CD47–treated animals compared with controls, indicating muscle pathology at the level of macrophages, we further analyzed that blocking CD47 attenuated muscle pathology (Fig. 5C). These an extended macrophage gene expression profile in these cells data suggest that Ab-mediated blockade of CD47 signaling to isolated from infected muscle. Thus, we used FACS to sort CD45+ macrophages in WT mice altered macrophage activity in ways CD11b+Ly6c+(hi and lo) macrophages from skeletal muscle of that were protective against TMEV-induced muscle pathology, as me/me and WT mice at 5 d P.I. for subsequent RNA analysis. In- seen in SHP-12/2 mice. terestingly, we observed significantly increased expression of vari- Because the CD47 receptor SIRPa is predominantly expressed on ous proinflammatory genes within sorted WT mouse macrophages macrophages (46, 51), macrophage phenotypic differences between compared with me/me mouse macrophages (Fig. 6A), including control and anti-CD47–treated mice were analyzed. Indeed, upon TNF-a, IL-1b, IL-6, caspase 1, iNOS, IRF-1, IRF-5, and IRF-8. immunofluorescence analysis of tissue sections, CD47-neutralized No significant differences in the expression of M2-associated mice displayed elevated Ly6c expression and reduced F4/80 ex- genes (arginase 1, FIZZ1 or Ym1) were observed between WT pression compared with controls (Fig. 5D), correlating with the and me/me macrophages (Fig. 6A). These data demonstrated that, immature phenotype of macrophages observed in me/me muscle although me/me mice display a much more pronounced infiltration (Fig. 4B, 4C). These data suggest that, in SHP-12/2 mice, mac- of macrophages into infected muscle, me/me mouse macrophages rophage functions that are normally controlled by CD47–SIRPa– were relatively immature compared with those in WT muscle, SHP-1 signaling inhibit extensive myofiber calcification and allow with the latter maturing into classically activated (M1) macro- continued muscle function. Alternatively or additionally, expression phages. Because no differences between M2-associated genes 2802 SHP-1 IN VIRUS-INDUCED MYOSITIS Downloaded from http://www.jimmunol.org/ by guest on September 26, 2021

FIGURE 4. Quantification and phenotypic analysis of muscle-infiltrating monocytes. (A) Schematic diagram depicting the typical maturation of blood- borne inflammatory monocytes into macrophages in inflamed tissues, demonstrating downregulation of CCR2 followed by downregulation of Ly6c and upregulation of F4/80. (B–D) WT and me/me mice were sacrificed at 5 d P.I., and cells were isolated from skeletal muscle for flow cytometry analysis. (B) Representative gating strategy of WT and me/me muscle depicting initial CD45+ gate (top panel) to exclude myofiber debris and nonhematopoietic cells, as well as CD11b+Ly6chi or CD11b+Ly6clo monocyte gates (middle panel). Absolute numbers of CD11b+Ly6chi or CD11b+Ly6clo cells/g of skeletal muscle quantified (n = 3), performed in triplicate (bottom panel). (C and D) F4/80 and CCR2 expression was evaluated in CD45+CD11b+Ly6clo and CD45+CD11b+ Ly6chi cells. (C) Representative contour plots of WT and me/me mice. F4/80 gate depicts F4/80hi cells, whereas CCR2 gate depicts CCR2+ cells. (D) CCR2 (upper panel) or F4/80 (lower panel) MFI levels were quantified from F4/80lo or CCR2lo populations, respectively, with corresponding maturation stages as shown in (A), (n = 3, performed in duplicate). *p , 0.05, ***p , 0.001. were observed between genotypes, these data support the idea cells following TMEV infection (40, 41), Interestingly, although that SHP-1 drives the maturation of muscle-infiltrating inflam- M1 genes were higher in WT mouse muscle macrophages com- matory monocytes into M1-like macrophages, likely contributing pared with me/me mouse muscle macrophages, these differences to inflammatory-mediated myofiber death and calcification. were either reversed or eliminated in CNS macrophages (Fig. 6B), Because the gene expression profile of WT muscle-infiltrating indicating that SHP-1 regulated macrophage maturation/ macrophages corresponded with our previous observations of differentiation within CNS tissue uniquely compared with that SHP-12/2 CNS-infiltrating macrophages (40, 41), we additionally seen in skeletal muscle. sorted spinal cord–infiltrating CD45hi cells at 5 d P.I. in the same mice and analyzed an extended gene profile in these cells. CNS- Depletion of macrophages reduces disease severity in WT mice infiltrating cells were gated on CD45hi, because we demonstrated Because our data suggested that inflammatory M1-like macro- previously that macrophages are the predominant CNS-infiltrating phages contribute to muscle damage and calcification in WT mice, The Journal of Immunology 2803 Downloaded from http://www.jimmunol.org/ by guest on September 26, 2021

FIGURE 5. Impact of CD47 neutralization on disease severity. WT BL/6 mice were infected with TMEV and injected with control or CD47-neutralizing Abs from 5 to 13 d P.I. (A) Mean clinical disease scores (6 SEM) of mice according to the following scale: 1, hunched posture and ruffled fur; 2, swollen muscles/abnormal gait; 3, one limb immobilized; 4, two or more limbs immobilized; and 5, death. (B) Muscle calcification observed upon dissection at 16 d P.I. Representative images of muscle calcification from uninjected and anti-CD47–injected mice (left panels); arrows indicate calcified areas. Represen- tative Alizarin red staining of muscle from uninjected and anti-CD47–injected mice (right panels) (original magnification 320). (C) Percentage of calcified area of muscle was measured by converting bright-field images of Alizarin red staining into 8-bit black and white files (left panels), measuring the area of dark staining (middle panels), and measuring the whole tissue area. Representative images of IgG-injected control and anti-CD47 Ab–injected animals are shown. Graph depicts the percentage calcified area (right panel). Uninjected and IgG-injected controls were pooled (n =7;n = 3 for CD47-neutralized animals). (D) Frozen muscle from control and CD47-neutralized animals at 16 d P.I. was labeled with Abs against Ly6c (left panels) and F4/80 (right panels) in red and stained with DAPI (blue). Representative images of untreated control and anti-CD47–injected mice (control n = 7, anti-CD47 n = 4). Scale bars, 100 mm. The experiment was performed in duplicate. *p , 0.05, **p , 0.01. we sought to determine whether depletion of macrophages could confirmed that macrophages are the predominant cells contribut- inhibit muscle calcification in these animals. Clodronate liposomes ing to TMEV-induced muscle calcification in WT animals. have been used extensively to selectively deplete macrophages in vivo (56–58). Thus, WT animals were injected with either TMEV replicates to high levels in M1 macrophages of WT mice control or clodronate liposomes every 3 d from 6 to 12 d P.I. and Because we had seen increased maturation of classically activated were sacrificed at 14 d P.I. Mice were assigned daily clinical macrophages in WT muscle compared with me/me muscle, it was disease scores from 8 to 14 d P.I. Animals receiving clodronate of further interest to characterize the replicative capacity of TMEV liposomes displayed significantly reduced clinical disease severity in this population, because it was reported that TMEV replicates in (Fig. 7A), suggesting that depletion of macrophages reduced mature macrophages but not in less mature lineage cells, including TMEV-induced muscle disease in WT mice. Upon dissection, all monocytes (59–61). At 5 and 9 d P.I., similar levels of TMEV five animals receiving control liposomes displayed moderate to Ag were found within myofibers of WT and SHP-12/2 mice severe signs of muscle calcification, whereas all five mice re- (Fig. 8A), indicating that TMEV infection of muscle occurred ceiving clodronate liposomes were relatively free of muscle cal- equally in these genotypes. Consistent with this observation, RNA cification (Fig. 7B, left panels), signifying that depletion of analysis of whole skeletal muscle tissue confirmed similar levels macrophages restricts TMEV-induced muscle calcification in WT of TMEV genomes in me/me and WT littermates at 5 d P.I., as mice. This was further demonstrated by closer inspection of the well as in mev/mev and WT littermates at 9 d P.I. (Fig. 8C). medial gastrocnemius muscle (Fig. 7B, middle panels) and by Nonetheless, immunofluorescent staining showed that some Ly6c+ alizarin red staining (Fig. 7B, right panels). Thus, these data cells were infected with TMEV (Fig. 8B), and these appeared 2804 SHP-1 IN VIRUS-INDUCED MYOSITIS Downloaded from

FIGURE 6. Gene expression in muscle or CNS-in- filtrating cells. (A) CD45+CD11b+Ly6c+ cells were sorted from skeletal muscle of WT and me/me litter- mates at 5 d P.I. (B) CD45+ cells were sorted from spinal cords of animals shown in (A). (A and B)RNA levels of IFN-b and M1/M2-associated genes were http://www.jimmunol.org/ measured using a multiplex bead-based assay. Gene expression levels were normalized to the mean of three housekeeping genes (HPRT1, TBP1, and GAPDH) for each sample (n = 3). *p , 0.05, **p , 0.01, ***p , 0.001, ****p , 0.0001. by guest on September 26, 2021

more prominent in muscle of WT mice compared with SHP-12/2 Discussion mice. To investigate this further, RNA analysis of muscle- The main phenotypic disease outcomes following peripheral infiltrating CD45+CD11b+Ly6c+ macrophages revealed many TMEV infection of suckling WT mice are extensive calcification more copies of TMEV genomes in muscle-infiltrating WT mouse and immobilization of limb skeletal muscles. Further, mice defi- macrophages compared with me/me mouse macrophages cient in SHP-1 on either of two different genetic backgrounds, (Fig. 8C). These data demonstrated that, although infection of while displaying equal levels of infection of skeletal muscle muscle fibers appeared to be SHP-1 independent, TMEV repli- compared with WT animals, never displayed limb muscle calci- cated preferentially in WT mouse macrophages compared with fication or immobilization. The present study was focused on me/me mouse macrophages. In sum, SHP-1 drove both increased understanding the basis for this dramatic phenotypic difference. M1 differentiation and increased virus replication in macrophages, First, we focused on the nature of ectopic calcification. In general, both of which were associated with increased inflammatory dis- ectopic calcification is defined as an inappropriate mineralization of ease in skeletal muscle of WT animals, as depicted schematically soft tissues and is divided into two classes: metastatic calcification in Fig. 9. occurs during a systemic mineral imbalance with elevated serum The Journal of Immunology 2805

FIGURE 7. Impact of macrophage depletion on disease severity. WT C3H mice were infected with TMEV and injected with control or clodronate liposomes from 6 to 12 d P.I. and sacrificed at 14 d P.I. (n = 5). (A) Mean clinical disease scores (6 SEM) of mice were calculated based on the following scale: 1, hunched posture and ruffled fur; 2, swollen muscles/abnormal gait; 3, one limb immobilized; 4, two or more limbs immobilized; and 5, death. (B) Representative images of muscle calcification observed upon dissection at 16 d P.I., arrows denote examples of calcified areas. Dissection images reveal calcification of whole animals (left panels), whereas zoomed images (middle panels) illustrate calcification of medial gastrocnemius muscle. Representative Downloaded from Alizarin red staining of gluteus maximus muscles (right panel) (original magnification 320). *p , 0.05. calcium and/or phosphorous levels, and dystrophic calcification vation that TMEV-induced calcification occurs within myofibers occurs in the absence of a systemic mineral imbalance and results (Fig. 2), the apparent fibrosis in skeletal muscle (Figs. 1, 2), the from tissue damage and cell death (62–64). Based on the obser- overwhelming tissue damage observed in infected muscles http://www.jimmunol.org/ by guest on September 26, 2021

FIGURE 8. TMEV replication in WT and SHP-12/2 muscle. (A)mev/mev mice and WT littermates were sacrificed at 5 or 9 d P.I., and muscle sections were labeled with Abs against TMEV (green) and stained with DAPI (blue) (n = 3). Sections from animals sacrificed at 5 d P.I. were labeled with Abs against F4/80 (red) (left panels). Scale bars, 50 mm. Sections from animals sacrificed at 9 d P.I. were labeled with Abs against Ly6c (red) (right panels). Scale bars, 100 mm. (B)mev/mev mice and WT littermates were sacrificed at 9 d P.I., and muscle sections were labeled with Abs against Ly6c (red) and TMEV (green) and stained with DAPI (blue); arrows denote infected Ly6c+ cells (n = 3). Scale bars, 50 mm. (C) TMEV RNA levels were quantified from CD45+CD11b+Ly6c+ cells sorted from C3H me/me and WT littermates at 5 d P.I. (me/me and C3H WT cells) and homogenized muscle of C3H me/me and WT littermates at 5 d P.I. (me/me and C3H WT muscle), as well as BL/6 mev/mev and WT littermates at 9 d P.I. (mev/mev and BL/6 WT muscle). n =3; C3H me/me and WT muscle, p = 0.1329; BL/6 mev/mev and WT muscle, p = 0.2890. **p , 0.01. 2806 SHP-1 IN VIRUS-INDUCED MYOSITIS Downloaded from http://www.jimmunol.org/

FIGURE 9. Proposed mechanism of SHP-1–mediated TMEV pathogenesis. Following TMEV infection of skeletal muscle, inflammatory monocytes (green) infiltrate skeletal muscle in WT and SHP-12/2 mice. In WT mice, monocytes differentiate into M1-like macrophages (red), supporting TMEV replication and contributing to muscle fiber damage and subsequent calcification (red circles), likely via proinflammatory cytokines (red lightning bolt) (upper panels). Following active replication, TMEV produces an abortive infection, inducing apoptosis of macrophages and limiting the numbers of in- filtrating cells. In the absence of SHP-1, abnormally high numbers of muscle-infiltrating monocytes remain immature (green), preventing efficient repli- cation of TMEV (lower panels). These immature monocyte-like cells do not undergo apoptosis and do not produce disease pathology in skeletal muscle.

(Figs. 1, 2), and the resemblance of calcified myofibers to those Our data suggest that the key immune regulator SHP-1 is by guest on September 26, 2021 from a murine model of Duchenne muscular dystrophy (mdx a primary factor driving dystrophic calcification in WT mice by mice) (65, 66), we concluded that TMEV causes dystrophic cal- promoting M1 maturation of inflammatory macrophages during cification of degenerating myofibers in WT mice. Thus, there is TMEV infection of skeletal muscle. Beyond promoting an in- likely to be a significant difference in either the production or flammatory M1 phenotype in WT macrophages, TMEV appeared removal of damaged myofibers that accounts for the high inci- to preferentially replicate in M1 macrophages in WT skeletal dence of calcified fibers in WT animals. muscle compared with non-M1 macrophages in me/me mice The finding that infection levels of muscle fibers were similar (Figs. 6, 8). As such, TMEV may use various cellular pathways to between genotypes (Fig. 8), the complete lack of calcified myo- its advantage in M1-differentiated macrophages as a strategy for fibers in me/me mice, and the observation that depletion of maintaining infection in the face of an acute inflammatory macrophages from WT mice reduced muscle calcification support response to the virus. The finding that TMEV replicates in terminally the idea that an increased rate of myofiber cell death mediated by differentiated macrophages, but not at earlier less-differentiated M1 macrophages in WT mice compared with me/me mice is re- stages, is consistent with previous studies, including those with sponsible for the increased muscle disease in WT animals. As TMEV (59–61, 70–81). In accord with these observations and the such, SHP-1–mediated macrophage skewing toward an M1 phe- finding that SHP-1 appeared to promote M1 differentiation, we notype in muscle may increase the expression of molecules det- propose that SHP-12/2 mouse macrophages are maintained at a less rimental to myofiber survival in virus-infected muscle, as differentiated state of development within infected skeletal muscle described in other disease models. For instance, many chronic and, therefore, do not provide suitable host factors for TMEV inflammatory conditions resulting from tissue damage or infection replication. are associated with cachexia, a process involving muscle wasting The data presented in this article demonstrate unanticipated roles (67). Mounting evidence has implicated a major role for proin- for SHP-1 in facilitating both the development of M1 macrophages flammatory cytokines, such as TNF-a, IL-1b, and IL-6, in pro- and clinical tissue pathology following virus infection. Initially, moting muscle wasting (67, 68). In particular, TNF-a was shown this observation was surprising, because SHP-1 was shown to limit to reduce myotube protein content, induce muscle fiber decay, M1 macrophage phenotype and extensive immune-mediated in- inhibit myogenic differentiation, and trigger apoptosis of myo- flammatory tissue damage in the CNS (40, 41, 45). Consistent with fibers, whereas IL-1b was reported to trigger inducible NO syn- this, production of inflammatory cytokines, proteolytic factors, and thase–mediated muscle fiber necrosis (67, 69). Thus, it is a strong NO by M1-like macrophages was demonstrated to promote possibility that increased levels of proinflammatory cytokines inflammatory demyelination (36). However, it also was reported secreted by M1 macrophages, such as TNF-a, IL-1b, and IL-6, in that SHP-1–deficient macrophages are defective with regard to muscles from TMEV-infected WT mice cause increased damage their ability to produce both IL-6 and IL-12 in response to TLR to myofibers or disrupt the muscle repair processes. stimulation, indicating that SHP-1 is a positive regulator of these The Journal of Immunology 2807

M1 responses (82). Thus, the function of SHP-1 in macrophages Disclosures may be context specific for which tissue factors play a deciding The authors have no financial conflicts of interest. role in the M1 skewing of macrophages and M1-mediated tissue damage. Therefore, the mechanisms by which tissue and pathogen-associated factors may influence the role of SHP-1 in References 1. Crum-Cianflone, N. F. 2008. Bacterial, fungal, parasitic, and viral myositis. Clin. macrophage differentiation need to be clarified. We are currently Microbiol. Rev. 21: 473–494. focused on deciphering these factors in skeletal muscle and CNS 2. Josselson, J., T. Pula, and J. H. Sadler. 1980. Acute rhabdomyolysis associated tissue in which TMEV infection produces an entirely different with an echovirus 9 infection. Arch. Intern. Med. 140: 1671–1672. 3. Jehn, U. W., and M. K. Fink. 1980. 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