Skeletal muscle interleukin 15 promotes CD8+ T-cell function and autoimmune myositis
© Huang et al. 2015
Received: 29 May 2015
Accepted: 8 September 2015
Published: 28 September 2015
Interleukin 15 (IL-15) is thought to be abundant in the skeletal muscle under steady state conditions based on RNA expression; however, the IL-15 RNA level may not reflect the protein level due to post-transcriptional regulation. Although exogenous protein treatment and overexpression studies indicated IL-15 functions in the skeletal muscle, how the skeletal muscle cell uses IL-15 remains unclear. In myositis patients, IL-15 protein is up-regulated in the skeletal muscle. Given the supporting role of IL-15 in CD8+ T-cell survival and activation and the pathogenic role of cytotoxic CD8+ T cells in polymyositis and inclusion-body myositis, we hypothesize that IL-15 produced by the inflamed skeletal muscle promotes myositis via CD8+ T cells.
Expression of IL-15 and IL-15 receptors at the protein level by skeletal muscle cells were examined under steady state and cytokine stimulation conditions. The functions of IL-15 in the skeletal muscle were investigated using Il15 knockout (Il15 −/− ) mice. The immune regulatory role of skeletal muscle IL-15 was determined by co-culturing cytokine-stimulated muscle cells and memory-like CD8+ T cells in vitro and by inducing autoimmune myositis in skeletal-muscle-specific Il15 −/− mice.
We found that the IL-15 protein was not expressed by skeletal muscle cells under steady state condition but induced by tumor necrosis factor alpha (TNF-α) and interferon gamma (IFN-γ) stimulation and expressed as IL-15/IL-15 receptor alpha (IL-15Rα) complex. Skeletal muscle cells expressed a scanty amount of IL-15 receptor beta (IL-15Rβ) under either conditions and only responded to a high concentration of IL-15 hyperagonist, but not IL-15. Consistently, deficiency of endogenous IL-15 affected neither skeletal muscle growth nor its responses to TNF-α and IFN-γ. On the other hand, the cytokine-stimulated skeletal muscle cells presented antigen and provided IL-15 to promote the effector function of memory-like CD8+ T cells. Genetic ablation of Il15 in skeletal muscle cells greatly ameliorated autoimmune myositis in mice.
These findings together indicate that skeletal muscle IL-15 directly regulates immune effector cells but not muscle cells and thus presents a potential therapeutic target for myositis.
KeywordsIL-15 Skeletal muscle CD8+ T cell Autoimmune myositis
Interleukin 15 (IL-15) is widely expressed as its high-affinity binding partner IL-15 receptor alpha (IL-15Rα) at the RNA level, and the expression of IL-15 is subjected to multiple post-transcriptional regulations . Newly synthesized IL-15 and IL-15Rα proteins form complex in the endoplasmic reticulum [2, 3]. The complex is transported to and displayed on the cell surface and used via the IL-15 receptor beta (IL-15Rβ) and the common gamma chain (γc) expressed on neighboring cells [4, 5]. The binding of IL-15/IL-15Rα complex to IL-15Rβ/γc on the responding cells triggers the activation of JAK-STAT3/5, phosphatidylinositol 3-kinase (PI3K)-AKT, and p42/44 mitogen-activated protein kinase (ERK) signaling pathways [6, 7]. This mode of IL-15 usage termed “trans-presentation” is essential for the development and homeostasis of memory CD8+ T cells, CD8αα+ intestinal intraepithelial T cells, and natural killer (NK) cells . Notably, all naturally produced IL-15 proteins detected in human and mouse serum and in activated dendritic cells are in complex with IL-15Rα [3, 9].
IL-15 has been thought of as a myokine due to the abundant mRNA expression in the skeletal muscle . Moreover, expression of Il15 mRNA is up-regulated along myoblast differentiation . Previous studies showed that exogenous treatment or overexpression of IL-15 promotes myoblast differentiation and muscle hypertrophy and ameliorates muscle wasting in cancer cachexia [12–16]. Whereas skeletal-muscle-specific overexpression or systemic infusion of IL-15 induces skeletal muscle atrophy in vivo [17–19]. Moreover, recent studies showed that exercise endurance is reduced in Il15 −/− mice and increased in skeletal-muscle-specific Il15-transgenic mice [18, 20]. However, the extensor digitorum longus (EDL) and soleus muscle isolated from these two types of genetically engineered mice show similar fatigue index ex vivo . Therefore, the function of IL-15 in the skeletal muscle under non-disease conditions remains elusive.
Inflammatory myopathies are a group of diseases that involve chronic muscle inflammation (myositis) accompanied with muscle weakness . The three main types including polymyositis, dermatomyositis, and inclusion body myositis are classified based on distinct clinicopathological features. They are idiopathic, but an autoimmune pathogenesis is strongly implicated. Dermatomyositis is mediated by complement, while polymyositis and inclusion-body myositis is mediated by CD8+ T cells that target major histocompatibility complex (MHC) class-I-expressing muscle cells through secreting cytotoxic effector molecules [23–25]. Interferon alpha (IFN-α), interferon gamma (IFN-γ), tumor necrosis factor alpha (TNF-α), and IL-1α/β are up-regulated in the muscle of myositis patients, which is implicated in the mediation of Th1 and pro-inflammatory responses . Stimulation of myoblasts with IL-1α/β, TNF-α, or IFN-γ induces IL-15 production in vitro [27, 28]. Consistently, elevation of IL-15 protein has been observed in the skeletal muscle of myositis patients [27, 28]. Local up-regulation of IL-15 in certain autoimmune diseases positively associates with disease severity. An increase of IL-15 protein in the intestinal mucosa and synovial cavity of celiac disease and rheumatoid arthritis patients, respectively, stimulates dendritic cells, NK cells, and effector T cells to exacerbate the disease [29, 30]. However, the role of skeletal muscle IL-15 in myositis has not been reported. Given the essential role of IL-15 in memory CD8+ T-cell survival and function and the pathogenic role of cytotoxic CD8+ T cells in polymyositis and inclusion-body myositis, we hypothesize that the skeletal muscle IL-15 promotes autoreactive CD8+ T-cell function, which contributes to the development of autoimmune myositis.
Considering that the level of IL-15 RNA may not reflect the level of protein due to post-transcriptional regulations  and that the usage of IL-15 by skeletal muscle cells has not been studied, we examined the expression of IL-15 and its receptors at the protein level in skeletal muscle cells under steady state and cytokine-stimulated conditions. We then examined the function of endogenous IL-15 in the skeletal muscle cell and its role in the development of autoimmune myositis.
C57BL/6J, B6.Cg-Tg(ACTA1-cre)79Jme/J (human alpha-skeletal actin (ACTA)-cre), and B6.129S1-synaptotagmin VII (Syt7) tm1Nan /J (Syt7 −/−) were purchased from The Jackson Laboratory (Bar Harbor, ME). Il15 −/− mice were purchased from Taconic and backcrossed to the C57BL/6J for at least 14 generations. Il15ra −/− mice were developed in our laboratory and backcrossed to the C57BL/6J for 27 generations . Il15 flox/flox (Il15 f/f ) mice were generated in our laboratory as previously described . Skeletal-muscle-specific Il15 −/− (ACTA-Il15 −/− ) mice were generated by crossing Il15 f/f with ACTA-cre mice. All experimental procedures were performed in accordance with protocols approved by the Institutional Animal Care and Use Committee of Academia Sinica.
Culture of skeletal muscle cells
Measurement of IL-15/IL-15Rα complex protein
Cells or skeletal muscle tissues were homogenized in non-denaturing cell lysis buffer (Cell signaling) containing protease inhibitor cocktail (Roche). For quantification of surface IL-15Rα-bound IL-15, muscle cells were incubated with acid glycine buffer as previously described . The amount of IL-15/IL-15Rα complex was measured by mouse IL-15/IL-15Rα Complex ELISA Kit (eBioscience).
To study signal transduction, adherent cells were washed, and a fixed volume of sodium dodecyl sulfate (SDS) sample buffer (50 mM Tris-Cl, pH 6.8, 6 % glycerol, 0.02 % bromophenol blue, 2 % SDS, and 2 % β-mercaptoethanol) was directly added to culture wells. Immunoblotting was performed using anti-p-STAT5 and t-STAT5 antibodies (Cell signaling) following the protocol of antibody manufacturer.
Gene expression analysis by quantitative real-time PCR (qPCR)
Total RNA was extracted by TRIzol, treated with DNase I, then reverse transcribed into cDNA by SuperScript III reverse transcriptase (Invitrogen, Life Technologies). Quantitative PCR was performed by Applied Biosystems 7500 Real-Time System using SYBR Green Master Mix. Relative gene expression levels were calculated by ABI 7500 software using acidic ribosomal phosphoprotein P0 (36B4) as the internal control. Primer pairs were pre-designed in PrimerBank  or designed by Primer Express software (Life Technologies) and are listed in Additional file 1: Table S1.
Co-culture of C2C12 cells with CD8+ T cells
The H-2K b (NM_001001892) coding sequence (nt. 77-1186) was cloned from the cDNA library of primary myotubes of C57BL/6J mice and then inserted into the EcoRI cloning site of lentiviral package plasmid pLKO AS3.1.EGFP3′. The full-length ovalbumin (OVA) cDNA with restriction enzyme cutting sites, 5′-NheI and 3′-EcoRI, was amplified by PCR from pcDNA3/OVA plasmid (kindly provided by Dr. Mi-Hua Tao, Academia Sinica) and inserted into lentiviral package plasmid pLAS2w.Ppuro. All lentiviral packaging plasmids and protocols were from the RNAi Core Facility, Academia Sinica. The EGFP-positive C2C12 myoblasts were sorted and subsequently infected with lentivirus-carrying OVA expression cassette and selected in growth medium containing 2 μg/ml puromycin (Sigma). The expression of H-2Kb and OVA was confirmed by flow cytometry and quantitative real-time PCR (qPCR), respectively. For CD8+ T cells, splenocytes of OT-1 mice were stimulated with OVA peptide as previously described  then cultured in medium containing IL-15 (30 ng/ml, eBioscience). After culturing for 8 days, more than 90 % of cells were CD8+CD44hiCD122hi, and these cells were used for experiments. C2C12 cells were treated with TNF-α and IFN-γ (1 ng/ml each) for 24 h. After washing with PBS, 24-h IL-15-deprived CD8+ T cells and 5 μg/ml brefeldin A (Sigma) were added simultaneously to C2C12 cells, followed by brief centrifugation to make the cell-cell contact. CD8+ T cells were harvested for intracellular cytokine analysis after co-culturing for 8 h.
Flow cytometry analysis
Lymphocyte surface markers were stained with antibodies against CD19 (6D5), H57 (H57-597), CD8 (53-6.7), CD44 (IM7), CD122 (TM-b1), and NK1.1 (PK136) (eBioscience and BioLegend). C2C12 cells were stained with biotin-conjugated antibodies against IL-15 (Cat. No. 500-P173Bt, PeproTech), IL-15Rα (Cat. No. BAF551, R&D), IL-15Rβ (CD122, clone: TM-b1, eBioscience), and γc (CD132, Cat. No. 554470, BD Biosciences) then incubated with APC-conjugated streptavidin (BD Biosciences). CD8+ T cells were stained with fixable viable dye eFluor506 (eBioscience) then intracellularly stained with antibodies against IFN-γ (XMG1.2, eBioscience) and granzyme B (NGZB, eBioscience). All data were acquired on LSRII (BD Biosciences) and analyzed by the FlowJo (Tree Star).
Induction of experimental autoimmune myositis
Preparation of mouse fast-type skeletal muscle myosin-binding protein C (C protein) fragment and induction of myositis were done as previously described with little modification . C protein fragment purified from Escherichia coli lysates was washed with 60 % isopropanol solution to remove endotoxin as previously described . Female mice, 8–10 weeks old, were intradermally immunized with 200 μg C protein emulsified in 200 μl complete Freund’s adjuvant (Sigma-Aldrich) at footpads, back, and tail base. Simultaneously, 0.5 μg pertussis toxin (Calbiochem) was intraperitoneally injected. The quadriceps muscle was harvested for histology and gene expression analysis 14 days after immunization. Each muscle block was cut into four sections with intervals of at least 200 μm. Histopathologic scoring was based on the most severe inflammation observed in the section among four sections and graded as previously described .
Results were represented as mean ± SEM. Statistical significance was determined by unpaired, two-tailed, Student’s t-test using GraphPad Prism 5 (GraphPad, San Diego, CA). p values less than 0.05 were considered significant.
Skeletal muscle cells express IL-15/IL-15Rα complex protein in response to TNF-α and IFN-γ stimulation
Previous studies found up-regulation of Il15 RNA during myoblast differentiation  and pro-inflammatory cytokine stimulation . Given the presence of post-transcriptional regulation of IL-15 expression and that all circulating IL-15 are in complex with IL-15Rα , we examined the expression of IL-15/IL-15Rα complex protein by skeletal muscle cell under the two conditions mentioned above. During a 6-day C2C12 myoblast-to-myotube differentiation, the cells showed a greater than 10-fold and a 3-fold increase of Il15 and Il15ra mRNA, respectively (Fig. 1a), whereas the cell lysate and culture medium contained no detectable IL-15/IL-15Rα protein by ELISA with a sensitivity of 3.9 pg/ml. This result indicates that although the level of Il15 and Il15ra mRNA increased along myoblast-to-myotube differentiation, there was little production of IL-15/IL-15Rα protein. We next examined whether pro-inflammatory cytokines induce IL-15 and IL-15Rα expression by skeletal muscle cells. We found that TNF-α and IFN-γ each up-regulated Il15 and Il15ra mRNA in C2C12 myotubes with distinct kinetics and together exerted a synergistic effect (Fig. 1b). A similar synergistic effect occurred in primary myotubes (Fig. 1c). TNF-α and IFN-γ treatment also up-regulated the expression of the IL-15/IL-15Rα complex protein in C2C12 myoblasts and myotubes (Fig. 1d) and in primary myotubes (Fig. 1e) in vitro. Most of the IL-15/IL-15Rα proteins were present in the C2C12 cell lysates (Fig. 1d), while nearly all IL-15/IL-15Rα proteins were present in the primary myotube lysate (Fig. 1e). Injection of TNF-α and IFN-γ into the quadriceps muscle in mice also greatly induced the expression of IL-15/IL-15Rα protein in the injected muscle (Fig. 1f).
IFN-α, another cytokine up-regulated in myositis muscle , induced Il15 and Il15ra mRNA and IL-15/IL-15Rα complex protein to the level similar to those induced by IFN-γ or TNF-α (Additional file 2: Figure S1). Other pro-inflammatory factors, such as IL-1α, IL-1β, and LPS, only transiently up-regulated Il15ra or Il15 mRNA but showed small effect on the induction of the IL-15/IL-15Rα protein (Additional file 2: Figure S1). Together, these results indicate that the expression of the IL-15/IL-15Rα complex protein by skeletal muscle cells was undetectable under steady state conditions, while induced by TNF-α, IFN-γ, or IFN-α that associates with Th1 response in myositis.
IL-15 is present on the surface of skeletal muscle cells
Skeletal muscle cells express a scanty amount of IL-15Rβ and only respond to a high concentration of IL-15 hyperagonist, but not IL-15
Due to the possibility of a very low level of IL-15Rβ expression, we examined whether IL-15 or an IL-15 hyperagonist induces signal transduction in C2C12 myotubes. The latter is a fusion protein of IL-15 and IL-15Rα-sushi domain, which possesses higher binding affinity for IL-15Rβ/γc (K d = 780 pM) and promotes a stronger proliferation of IL-15Rβ/γc-bearing cells (EC50 = 25 pM) than IL-15 (K d = 13.5 nM, EC50 = 3 nM) . We found that IL-15 did not induce STAT5 phosphorylation at a concentration up to 400 ng/ml (26.7 nM) (Fig. 3c). Whereas the IL-15 hyperagonist induced moderate but significant STAT5 phosphorylation at concentrations of 100 ng/ml (3.4 nM) and higher (Fig. 3c), which are much higher than that required for the proliferation of IL-15Rβ/γc-bearing cells (EC50 = 25 pM) and for STAT5 phosphorylation in pre-activated murine CD8+ cells (EC50 = 10 pM) [40, 41]. In addition to STAT5 phosphorylation, IL-15 and IL-15 hyperagonist did not induce phosphorylation of STAT3, AKT, and ERK in skeletal muscle cells (data not shown). The IL-15 hyperagonist-induced STAT5 phosphorylation was completely blocked by the IL-15Rβ-blocking antibody TM-b1 (Fig. 3d). However, neither IL-15 nor IL-15 hyperagonist induced STAT5 phosphorylation in C2C12 myotubes pre-treated with TNF-α and IFN-γ (Fig. 3e), which is in line with the decrease of Il15rb mRNA after cytokine treatment (Fig. 3b). Consistent with the STAT5 phosphorylation results (Fig. 3c), 100 ng/ml of IL-15 hyperagonist, but not IL-15, induced atrophy of C2C12 myotubes (Fig. 3f). In summary, C2C12 and primary skeletal muscle cells expressed a scanty IL-15Rβ under steady state condition and TNF-α/IFN-γ stimulation and only responded to a high concentration of IL-15 hyperagonist, but not IL-15. Moreover, we found that Il15 −/− mice showed normal skeletal muscle mass (Additional file 3: Figure S2), myoblast differentiation (Additional file 4: Figure S3), cardiotoxin-induced muscle regeneration (Additional file 5: Figure S4), and compensatory hypertrophy of plantaris muscle (Additional file 6: Figure S5). The in vitro and in vivo results collectively suggest that the skeletal muscle cells do not use IL-15.
IL-15 deficiency does not affect the response of primary myotube to TNF-α and IFN-γ stimulation
Skeletal muscle cells stimulated with TNF-α and IFN-γ present antigen and provide IL-15 to memory-like CD8+ T cells
Skeletal muscle IL-15 promotes the progression of autoimmune myositis
As two previously reported autoimmune myositis models, wt mice immunized with C protein [38, 53] and Syt7 −/− mice [54, 55], did not develop myositis in our hand, we immunized mice of Syt7 −/− background with C protein because impaired muscle membrane sealing due to Syt7 deficiency facilitates myositis induction [54, 56]. Similar to previously reported pathology in autoimmune myositis, we found that C-protein-immunized Syt7 −/− Il15 f/f mice developed mononuclear cell infiltration (Fig. 6d). The mononuclear cells predominantly infiltrated into the endomysium as well as the perimysium and perivascular region (Fig. 6d (left)). The immunized Syt7 −/− Il15 f/f mice also developed focal lymphatic invasion of muscle fibers that features CD8+ T-cell-mediated myositis (Fig. 6d (middle)). Whereas C-protein-immunized Syt7 −/− ACTA-Il15 −/− mice showed a significantly reduced mononuclear cell infiltration and histopathology score (Fig. 6d). We also found elevation of Cd4, Cd8α, and F4/80 mRNA in the skeletal muscle of the immunized Syt7 −/− Il15 f/f mice (Fig. 6e), suggesting infiltration of CD4+ and CD8+ T cells and macrophages. The expression of MHC class I subunit B2m; pro-inflammatory cytokines Tnfa, Ifng, and Il1β: and effector molecule Prf1 were also induced in the skeletal muscle of the immunized Syt7 −/− Il15 f/f mice (Fig. 6f). Whereas all these molecules examined were not induced in the C-protein-immunized Syt7 −/− ACTA-Il15 −/− mice (Fig. 6e, f). These results together demonstrate that genetic ablation of skeletal muscle IL-15 greatly reduced the pathogenesis of autoimmune myositis.
In this study, we examined the expression and function of the skeletal muscle cell IL-15. We found that the IL-15/IL-15Rα protein was not produced by skeletal muscle cells under steady state conditions but highly induced by TNF-α and IFN-γ and presented on the cell surface. Rather than being used by skeletal muscle cells, the IL-15 directly promoted the effector function of memory-like CD8+ T cells in vitro and exacerbated the progression of autoimmune myositis in vivo. These results suggest that the endogenous IL-15 of the skeletal muscle cell functions as an immune regulator in an inflammatory skeletal muscle microenvironment.
IL-15 has been reported to affect skeletal muscle physiology, but IL-15 signaling in skeletal muscle cells remains unclear. Our findings shed some light on this. First, C2C12 and primary muscle cells expressed very low levels of Il15rb mRNA and undetectable levels of cell surface IL-15Rβ (Fig. 3a, b). Second, a soluble IL-15 hyperagonist, but not IL-15, induced STAT5 phosphorylation in C2C12 myotube at 100 ng/ml and above (Fig. 3c). These concentrations are much higher than that required for binding to IL-15Rβ/γc and the pg/ml level of circulating the IL-15/IL-15Rα complex , which suggests that the myotubes do not use soluble IL-15 or IL-15/IL-15Rα complex under steady state conditions. Consistently, our in vivo studies showed that IL-15 deficiency did not affect skeletal muscle mass, cardiotoxin-induced muscle regeneration, and compensatory hypertrophy of plantaris muscle (Additional file 3, 5, and 6: Figure S2, S4, and S5). Although trans-presentation of cell-bound IL-15/IL-15Rα among the stimulated muscle cells may reach a high enough local concentration to trigger signaling through the sparsely expressed IL-15Rβ/γc, Il15 knockout did not affect the response of the primary myotube to TNF-α and IFN-γ in vitro (Fig. 4). Collectively, our results support that the skeletal muscle cells do not use IL-15 for skeletal muscle growth, regeneration, or inflammatory responses.
Our in vitro results appear different from previous in vitro studies showing that exogenous IL-15 promotes skeletal muscle hypertrophy [12, 14]. The difference may partly result from the intrinsic differences between the C2C12 and C2 cell lines used in this and previous studies, respectively. C2C12 cells have a higher differentiation potency and insulin-like growth factor 1 (IGF-I) level than C2 cells . IGF-I is a strong stimulator for muscle hypertrophy . An earlier study indicates that IL-15-induced C2 differentiation was only revealed in the absence of IGF-I signaling . Therefore, it is possible that the higher level of IGF-I masked IL-15-induced hypertrophy in C2C12 cells under steady state condition. However, IL-15 hyperagonist did not induce STAT5 phosphorylation in TNF-α and IFN-γ-pretreated myotubes (Fig. 3e), in which 90 % of endogenous IGF-I was downregulated . Therefore, the scanty amount of IL-15Rβ likely limits the use of IL-15 by the muscle cells.
Although various IL-15 functions in the skeletal muscle were reported previously, some controversial results exist between those in vitro and in vivo studies. In contrast to muscle hypertrophy induced by IL-15 in vitro, muscle atrophy was observed in mice carrying skeletal-muscle-specific IL-15 transgene  or receiving systemic infusion of exogenous IL-15 . The muscle atrophy may be contributed by fatal leukemia  and metabolic dysregulation  induced by overexpression of IL-15 . In addition, recent studies show that exercise endurance increases in skeletal-muscle-specific Il15-transgenic mice and reduces in Il15 −/− mice [18, 20], despite that their earlier ex vivo study found no difference in the fatigue index between the EDL or soleus muscle isolated from Il15 −/− mice and skeletal-muscle-specific Il15-transgenic mice . Collectively, the inconsistency between the in vitro/ex vivo and in vivo results suggests that the change of muscle mass or exercise endurance in vivo is not caused by the direct effect of IL-15 on the skeletal muscle.
IL-15 has been reported to be involved in a number of autoimmune diseases, including rheumatoid arthritis, inflammatory bowel disease, and multiple sclerosis, in which IL-15 promotes the effector function of cytotoxic CD8+ T cells to destroy the target tissues . Considering that TNF-α and IFN-γ induced the expression of IL-15/IL-15Rα and T-cell interacting molecules in skeletal muscle cells, the stimulated muscle cells may directly communicate with T cells. Indeed, we clearly demonstrated in vitro that the stimulated skeletal muscle cells presented antigen and IL-15 to memory-like CD8+ T cells and enhanced their effector function. Following induction of autoimmune myositis in vivo, skeletal-muscle-specific Il15 −/− mice showed reduced mononuclear cell infiltration, histopathology score, and expression of inflammatory molecules. Our data together suggest a scenario in which skeletal muscle IL-15 promotes the production of cytotoxic molecules, such as granzyme, by memory CD8+ T cells to necrotize muscle cells, which triggers phagocyte recruitment and inflammation. The locally enriched cytokines, TNF-α and IFN-γ (Fig. 6f), may stimulate IL-15 expression by muscle cells to form a feed-forward loop to perpetuate the inflammatory milieu, which contributes to the progression of autoimmune myositis.
We provide new insights into the function of skeletal muscle IL-15. Rather than being used by the muscle cell itself, the skeletal muscle IL-15 directly promotes the effector function of memory-like CD8+ T cells, which facilitates the formation of a pro-inflammatory skeletal muscle microenvironment during myositis progression. Given that IL-15 is not required for muscle growth and regeneration, IL-15 has the potential to be a suitable therapeutic target for autoimmune myositis.
- ACTA :
human alpha-skeletal actin
extensor digitorum longus muscle
interleukin 15 receptor alpha
myosin heavy chain
tibialis anterior muscle
- γc :
common gamma chain
We thank the Taiwan Mouse Clinic (MOST 103-2325-B-001-015), funded by the National Research Program for Biopharmaceuticals at the Ministry of Science and Technology (MOST) of Taiwan, for technical support in histology experiments. We also acknowledge the assistance of FACS Facility, Transgenic Core Facility, Genomics Core Facility, Imaging Core Facility, and Scientific English Editing Core Facility at the Institute of Molecular Biology. This study was supported by MOST (NSC 98-2320-B-001-004-MY3) and Academia Sinica, Taiwan.
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- Budagian V, Bulanova E, Paus R, Bulfone-Paus S. IL-15/IL-15 receptor biology: a guided tour through an expanding universe. Cytokine Growth Factor Rev. 2006;17(4):259–80. doi:10.1016/j.cytogfr.2006.05.001.View ArticlePubMedGoogle Scholar
- Duitman EH, Orinska Z, Bulanova E, Paus R, Bulfone-Paus S. How a cytokine is chaperoned through the secretory pathway by complexing with its own receptor: lessons from interleukin-15 (IL-15)/IL-15 receptor alpha. Mol Cell Biol. 2008;28(15):4851–61. doi:10.1128/mcb.02178-07.PubMed CentralView ArticlePubMedGoogle Scholar
- Mortier E, Woo T, Advincula R, Gozalo S, Ma A. IL-15Ralpha chaperones IL-15 to stable dendritic cell membrane complexes that activate NK cells via trans presentation. J Exp Med. 2008;205(5):1213–25. doi:10.1084/jem.20071913.PubMed CentralView ArticlePubMedGoogle Scholar
- Stonier SW, Schluns KS. Trans-presentation: a novel mechanism regulating IL-15 delivery and responses. Immunol Lett. 2010;127(2):85–92. doi:10.1016/j.imlet.2009.09.009.PubMed CentralView ArticlePubMedGoogle Scholar
- Waldmann TA. The biology of interleukin-2 and interleukin-15: implications for cancer therapy and vaccine design. Nat Rev Immunol. 2006;6(8):595–601. doi:10.1038/nri1901.View ArticlePubMedGoogle Scholar
- Ellery JM, Nicholls PJ. Alternate signalling pathways from the interleukin-2 receptor. Cytokine Growth Factor Rev. 2002;13(1):27–40.View ArticlePubMedGoogle Scholar
- Marzec M, Halasa K, Kasprzycka M, Wysocka M, Liu X, Tobias JW, et al. Differential effects of interleukin-2 and interleukin-15 versus interleukin-21 on CD4+ cutaneous T-cell lymphoma cells. Cancer Res. 2008;68(4):1083–91. doi:10.1158/0008-5472.can-07-2403.View ArticlePubMedGoogle Scholar
- Fehniger TA, Caligiuri MA. Interleukin 15: biology and relevance to human disease. Blood. 2001;97(1):14–32.View ArticlePubMedGoogle Scholar
- Bergamaschi C, Bear J, Rosati M, Beach RK, Alicea C, Sowder R, et al. Circulating IL-15 exists as heterodimeric complex with soluble IL-15Ralpha in human and mouse serum. Blood. 2012;120(1):e1–8. doi:10.1182/blood-2011-10-384362.PubMed CentralView ArticlePubMedGoogle Scholar
- Grabstein KH, Eisenman J, Shanebeck K, Rauch C, Srinivasan S, Fung V, et al. Cloning of a T cell growth factor that interacts with the beta chain of the interleukin-2 receptor. Science. 1994;264(5161):965–8.View ArticlePubMedGoogle Scholar
- Quinn LS, Strait-Bodey L, Anderson BG, Argiles JM, Havel PJ. Interleukin-15 stimulates adiponectin secretion by 3 T3-L1 adipocytes: evidence for a skeletal muscle-to-fat signaling pathway. Cell Biol Int. 2005;29(6):449–57. doi:10.1016/j.cellbi.2005.02.005.View ArticlePubMedGoogle Scholar
- Quinn LS, Anderson BG, Drivdahl RH, Alvarez B, Argiles JM. Overexpression of interleukin-15 induces skeletal muscle hypertrophy in vitro: implications for treatment of muscle wasting disorders. Exp Cell Res. 2002;280(1):55–63.View ArticlePubMedGoogle Scholar
- Quinn LS, Haugk KL, Damon SE. Interleukin-15 stimulates C2 skeletal myoblast differentiation. Biochem Biophys Res Commun. 1997;239(1):6–10. doi:10.1006/bbrc.1997.7414.View ArticlePubMedGoogle Scholar
- Quinn LS, Haugk KL, Grabstein KH. Interleukin-15: a novel anabolic cytokine for skeletal muscle. Endocrinology. 1995;136(8):3669–72.PubMedGoogle Scholar
- Harcourt LJ, Holmes AG, Gregorevic P, Schertzer JD, Stupka N, Plant DR, et al. Interleukin-15 administration improves diaphragm muscle pathology and function in dystrophic mdx mice. Am J Pathol. 2005;166(4):1131–41. doi:10.1016/s0002-9440(10)62333-4.PubMed CentralView ArticlePubMedGoogle Scholar
- Carbo N, Lopez-Soriano J, Costelli P, Busquets S, Alvarez B, Baccino FM, et al. Interleukin-15 antagonizes muscle protein waste in tumour-bearing rats. Br J Cancer. 2000;83(4):526–31. doi:10.1054/bjoc.2000.1299.PubMed CentralView ArticlePubMedGoogle Scholar
- Quinn LS, Anderson BG, Strait-Bodey L, Stroud AM, Argiles JM. Oversecretion of interleukin-15 from skeletal muscle reduces adiposity. Am J Physiol Endocrinol Metab. 2009;296(1):E191–202. doi:10.1152/ajpendo.90506.2008.PubMed CentralView ArticlePubMedGoogle Scholar
- Quinn LS, Anderson BG, Conner JD, Wolden-Hanson T. IL-15 overexpression promotes endurance, oxidative energy metabolism, and muscle PPARdelta, SIRT1, PGC-1alpha, and PGC-1beta expression in male mice. Endocrinology. 2013;154(1):232–45. doi:10.1210/en.2012-1773.PubMed CentralView ArticlePubMedGoogle Scholar
- Pistilli EE, Alway SE. Systemic elevation of interleukin-15 in vivo promotes apoptosis in skeletal muscles of young adult and aged rats. Biochem Biophys Res Commun. 2008;373(1):20–4. doi:10.1016/j.bbrc.2008.05.188.PubMed CentralView ArticlePubMedGoogle Scholar
- Quinn LS, Anderson BG, Conner JD, Wolden-Hanson T, Marcell TJ. IL-15 is required for postexercise induction of the pro-oxidative mediators PPARdelta and SIRT1 in male mice. Endocrinology. 2014;155(1):143–55. doi:10.1210/en.2013-1645.View ArticlePubMedGoogle Scholar
- Pistilli EE, Bogdanovich S, Garton F, Yang N, Gulbin JP, Conner JD, et al. Loss of IL-15 receptor alpha alters the endurance, fatigability, and metabolic characteristics of mouse fast skeletal muscles. J Clin Invest. 2011;121(8):3120–32. doi:10.1172/jci44945.PubMed CentralView ArticlePubMedGoogle Scholar
- Dalakas MC. Immunotherapy of myositis: issues, concerns and future prospects. Nat Rev Rheumatol. 2010;6(3):129–37. doi:10.1038/nrrheum.2010.2.View ArticlePubMedGoogle Scholar
- Dalakas MC. Pathogenesis and therapies of immune-mediated myopathies. Autoimmun Rev. 2012;11(3):203–6. doi:10.1016/j.autrev.2011.05.013.View ArticlePubMedGoogle Scholar
- Malmstrom V, Venalis P, Albrecht I. T cells in myositis. Arthritis Res Ther. 2012;14(6):230. doi:10.1186/ar4116.PubMed CentralView ArticlePubMedGoogle Scholar
- Zong M, Lundberg IE. Pathogenesis, classification and treatment of inflammatory myopathies. Nat Rev Rheumatol. 2011;7(5):297–306. doi:10.1038/nrrheum.2011.39.View ArticlePubMedGoogle Scholar
- De Paepe B, Creus KK, De Bleecker JL. Role of cytokines and chemokines in idiopathic inflammatory myopathies. Curr Opin Rheumatol. 2009;21(6):610–6. doi:10.1097/BOR.0b013e3283317b31.View ArticlePubMedGoogle Scholar
- Sugiura T, Harigai M, Kawaguchi Y, Takagi K, Fukasawa C, Ohsako-Higami S, et al. Increased IL-15 production of muscle cells in polymyositis and dermatomyositis. Int Immunol. 2002;14(8):917–24.View ArticlePubMedGoogle Scholar
- Sugiura T, Kawaguchi Y, Harigai M, Takagi K, Ohta S, Fukasawa C, et al. Increased CD40 expression on muscle cells of polymyositis and dermatomyositis: role of CD40-CD40 ligand interaction in IL-6, IL-8, IL-15, and monocyte chemoattractant protein-1 production. J Immunol. 2000;164(12):6593–600.View ArticlePubMedGoogle Scholar
- Abadie V, Jabri B. IL-15: a central regulator of celiac disease immunopathology. Immunol Rev. 2014;260(1):221–34. doi:10.1111/imr.12191.PubMed CentralView ArticlePubMedGoogle Scholar
- McInnes IB, Schett G. Cytokines in the pathogenesis of rheumatoid arthritis. Nat Rev Immunol. 2007;7(6):429–42. doi:10.1038/nri2094.View ArticlePubMedGoogle Scholar
- Waldmann TA, Tagaya Y. The multifaceted regulation of interleukin-15 expression and the role of this cytokine in NK cell differentiation and host response to intracellular pathogens. Annu Rev Immunol. 1999;17:19–49. doi:10.1146/annurev.immunol.17.1.19.View ArticlePubMedGoogle Scholar
- Chang CL, Lai YG, Hou MS, Huang PL, Liao NS. IL-15Ralpha of radiation-resistant cells is necessary and sufficient for thymic invariant NKT cell survival and functional maturation. J Immunol. 2011;187(3):1235–42. doi:10.4049/jimmunol.1100270.View ArticlePubMedGoogle Scholar
- Liou YH, Wang SW, Chang CL, Huang PL, Hou MS, Lai YG, et al. Adipocyte IL-15 regulates local and systemic NK cell development. J Immunol. 2014;193(4):1747–58. doi:10.4049/jimmunol.1400868.View ArticlePubMedGoogle Scholar
- Blanco-Bose WE, Yao CC, Kramer RH, Blau HM. Purification of mouse primary myoblasts based on alpha 7 integrin expression. Exp Cell Res. 2001;265(2):212–20. doi:10.1006/excr.2001.5191.View ArticlePubMedGoogle Scholar
- Dubois S, Mariner J, Waldmann TA, Tagaya Y. IL-15Ralpha recycles and presents IL-15 in trans to neighboring cells. Immunity. 2002;17(5):537–47.View ArticlePubMedGoogle Scholar
- Spandidos A, Wang X, Wang H, Seed B. PrimerBank: a resource of human and mouse PCR primer pairs for gene expression detection and quantification. Nucleic Acids Res. 2010;38(Database issue):D792–9. doi:10.1093/nar/gkp1005.PubMed CentralView ArticlePubMedGoogle Scholar
- Pulle G, Vidric M, Watts TH. IL-15-dependent induction of 4-1BB promotes antigen-independent CD8 memory T cell survival. J Immunol. 2006;17(5):2739–48.View ArticleGoogle Scholar
- Sugihara T, Sekine C, Nakae T, Kohyama K, Harigai M, Iwakura Y, et al. A new murine model to define the critical pathologic and therapeutic mediators of polymyositis. Arthritis Rheum. 2007;56(4):1304–14. doi:10.1002/art.22521.View ArticlePubMedGoogle Scholar
- Linares D, Echevarria I, Mana P. Single-step purification and refolding of recombinant mouse and human myelin oligodendrocyte glycoprotein and induction of EAE in mice. Protein Expr Purif. 2004;34(2):249–56. doi:10.1016/j.pep.2003.11.016.View ArticlePubMedGoogle Scholar
- Mortier E, Quemener A, Vusio P, Lorenzen I, Boublik Y, Grotzinger J, et al. Soluble interleukin-15 receptor alpha (IL-15R alpha)-sushi as a selective and potent agonist of IL-15 action through IL-15R beta/gamma. Hyperagonist IL-15 x IL-15R alpha fusion proteins. J Biol Chem. 2006;281(3):1612–9. doi:10.1074/jbc.M508624200.View ArticlePubMedGoogle Scholar
- Ring AM, Lin JX, Feng D, Mitra S, Rickert M, Bowman GR, et al. Mechanistic and structural insight into the functional dichotomy between IL-2 and IL-15. Nat Immunol. 2012;13(12):1187–95. doi:10.1038/ni.2449.PubMed CentralView ArticlePubMedGoogle Scholar
- Acharyya S, Ladner KJ, Nelsen LL, Damrauer J, Reiser PJ, Swoap S, et al. Cancer cachexia is regulated by selective targeting of skeletal muscle gene products. J Clin Invest. 2004;114(3):370–8. doi:10.1172/jci20174.PubMed CentralView ArticlePubMedGoogle Scholar
- Guttridge DC, Mayo MW, Madrid LV, Wang CY, Baldwin Jr AS. NF-kappaB-induced loss of MyoD messenger RNA: possible role in muscle decay and cachexia. Science. 2000;289(5488):2363–6.View ArticlePubMedGoogle Scholar
- Agostini C, Zambello R, Facco M, Perin A, Piazza F, Siviero M, et al. CD8 T-cell infiltration in extravascular tissues of patients with human immunodeficiency virus infection. Interleukin-15 upmodulates costimulatory pathways involved in the antigen-presenting cells-T-cell interaction. Blood. 1999;93(4):1277–86.PubMedGoogle Scholar
- Gil M, Park SJ, Chung YS, Park CS. Interleukin-15 enhances proliferation and chemokine secretion of human follicular dendritic cells. Immunology. 2010;130(4):536–44. doi:10.1111/j.1365-2567.2010.03252.x.PubMed CentralView ArticlePubMedGoogle Scholar
- Perera LP, Goldman CK, Waldmann TA. IL-15 induces the expression of chemokines and their receptors in T lymphocytes. J Immunol. 1999;162(5):2606–12.PubMedGoogle Scholar
- Tourkova IL, Shurin GV, Chatta GS, Perez L, Finke J, Whiteside TL, et al. Restoration by IL-15 of MHC class I antigen-processing machinery in human dendritic cells inhibited by tumor-derived gangliosides. J Immunol. 2005;175(5):3045–52.View ArticlePubMedGoogle Scholar
- Tourkova IL, Yurkovetsky ZR, Gambotto A, Makarenkova VP, Perez L, Balkir L, et al. Increased function and survival of IL-15-transduced human dendritic cells are mediated by up-regulation of IL-15Ralpha and Bcl-2. J Leukoc Biol. 2002;72(5):1037–45.PubMedGoogle Scholar
- Marino M, Scuderi F, Provenzano C, Bartoccioni E. Skeletal muscle cells: from local inflammatory response to active immunity. Gene Ther. 2011;18(2):109–16. doi:10.1038/gt.2010.124.View ArticlePubMedGoogle Scholar
- Pavlath GK. Regulation of class I MHC expression in skeletal muscle: deleterious effect of aberrant expression on myogenesis. J Neuroimmunol. 2002;125(1–2):42–50.View ArticlePubMedGoogle Scholar
- Williams DB, Barber BH, Flavell RA, Allen H. Role of beta 2-microglobulin in the intracellular transport and surface expression of murine class I histocompatibility molecules. J Immunol. 1989;142(8):2796–806.PubMedGoogle Scholar
- Figarella-Branger D, Civatte M, Bartoli C, Pellissier JF. Cytokines, chemokines, and cell adhesion molecules in inflammatory myopathies. Muscle Nerve. 2003;28(6):659–82. doi:10.1002/mus.10462.View ArticlePubMedGoogle Scholar
- Sugihara T, Okiyama N, Suzuki M, Kohyama K, Matsumoto Y, Miyasaka N, et al. Definitive engagement of cytotoxic CD8 T cells in C protein-induced myositis, a murine model of polymyositis. Arthritis Rheum. 2010;62(10):3088–92. doi:10.1002/art.27625.View ArticlePubMedGoogle Scholar
- Chakrabarti S, Kobayashi KS, Flavell RA, Marks CB, Miyake K, Liston DR, et al. Impaired membrane resealing and autoimmune myositis in synaptotagmin VII-deficient mice. J Cell Biol. 2003;162(4):543–9. doi:10.1083/jcb.200305131.PubMed CentralView ArticlePubMedGoogle Scholar
- Young NA, Sharma R, Friedman AK, Kaffenberger BH, Bolon B, Jarjour WN. Aberrant muscle antigen exposure in mice is sufficient to cause myositis in a Treg cell-deficient milieu. Arthritis Rheum. 2013;65(12):3259–70. doi:10.1002/art.38184.PubMed CentralView ArticlePubMedGoogle Scholar
- Han R. Muscle membrane repair and inflammatory attack in dysferlinopathy. Skeletal Muscle. 2011;1(1):10. doi:10.1186/2044-5040-1-10.PubMed CentralView ArticlePubMedGoogle Scholar
- Sharples AP, Al-Shanti N, Stewart CE. C2 and C2C12 murine skeletal myoblast models of atrophic and hypertrophic potential: relevance to disease and ageing? J Cell Physiol. 2010;225(1):240–50. doi:10.1002/jcp.22252.View ArticlePubMedGoogle Scholar
- Rommel C, Bodine SC, Clarke BA, Rossman R, Nunez L, Stitt TN, et al. Mediation of IGF-1-induced skeletal myotube hypertrophy by PI(3)K/Akt/mTOR and PI(3)K/Akt/GSK3 pathways. Nat Cell Biol. 2001;3(11):1009–13. doi:10.1038/ncb1101-1009.View ArticlePubMedGoogle Scholar
- Fernandez-Celemin L, Pasko N, Blomart V, Thissen JP. Inhibition of muscle insulin-like growth factor I expression by tumor necrosis factor-alpha. Am J Physiol Endocrinol Metab. 2002;283(6):E1279–90. doi:10.1152/ajpendo.00054.2002.View ArticlePubMedGoogle Scholar
- Fehniger TA, Suzuki K, Ponnappan A, VanDeusen JB, Cooper MA, Florea SM, et al. Fatal leukemia in interleukin 15 transgenic mice follows early expansions in natural killer and memory phenotype CD8+ T cells. J Exp Med. 2001;193(2):219–31.PubMed CentralView ArticlePubMedGoogle Scholar
- Barra NG, Chew MV, Reid S, Ashkar AA. Interleukin-15 treatment induces weight loss independent of lymphocytes. PLoS One. 2012;7(6):e39553. doi:10.1371/journal.pone.0039553.PubMed CentralView ArticlePubMedGoogle Scholar
- Walter U, Santamaria P. CD8+ T cells in autoimmunity. Curr Opin Immunol. 2005;17(6):624–31. doi:10.1016/j.coi.2005.09.014.View ArticlePubMedGoogle Scholar