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Skeletal muscle interleukin 15 promotes CD8(+) T-cell function and autoimmune myositis.

Huang PL, Hou MS, Wang SW, Chang CL, Liou YH, Liao NS - Skelet Muscle (2015)

Bottom Line: 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.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.

View Article: PubMed Central - PubMed

Affiliation: Molecular Cell Biology, Taiwan International Graduate Program, Institute of Molecular Biology, Academia Sinica, and Graduate Institute of Life Sciences, National Defense Medical Center, Taipei, Taiwan ; Institute of Molecular Biology, Academia Sinica, Taipei, 11529 Taiwan.

ABSTRACT

Background: 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.

Methods: 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.

Results: 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.

Conclusions: 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.

No MeSH data available.


Related in: MedlinePlus

High concentration of IL-15 hyperagonist, but not IL-15, induces STAT5 signaling and atrophy in skeletal muscle cells. a Analysis of IL-15Rβ and γc expression on C2C12 myoblasts under the same condition of Fig. 2a. Data are representative of three independent experiments with similar results. b Expression of Il15rb and γc mRNA in C2C12 and primary myotube. Samples were collected 24 h after cytokine treatment and analyzed by qPCR. Data are triplicates and representative of two independent experiments with similar results. c Immunoblotting of STAT5 phosphorylation in C2C12 myotubes after treating with IL-15 or IL-15 hyperagonist for 30 min. Quantification data of four independent experiments are shown below. d Anti-IL-15Rβ antibody diminished IL-15 hyperagonist-induced STAT5 phosphorylation in C2C12 myotubes. Myotubes were pretreated with anti-IL-15Rβ or isotype control antibody for 1 h and then treated with IL-15 hyperagonist for 30 min. Data are representative of two independent experiments with similar results. e Immunoblotting of STAT5 phosphorylation in C2C12 myotubes that were first stimulated with TNF-α and IFN-γ for 24 h and then treated with exogenous IL-15 or IL-15 hyperagonist for 30 min. Data are representative of two independent experiments with similar results. f Accumulation of myosin heavy chain (MyHC) in C2C12 myotubes treated with IL-15 (50 and 100 ng/ml), IL-15 hyperagonist (50 and 100 ng/ml), or vehicle (0 ng/ml). After 48 h, cells were immunostained with FITC-labeled anti-myosin antibody to evaluate the accumulation of MyHC. MyHC-positive area per microscopic field are shown in the right panel and represented as the percentages of con. Quantification data were pooled from three independent experiments. Each symbol represents the quantification data from one microscopic field. Scale bar = 500 μm. Data are mean ± SEM. *p < 0.05, **p < 0.01, ***p < 0.001
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Fig3: High concentration of IL-15 hyperagonist, but not IL-15, induces STAT5 signaling and atrophy in skeletal muscle cells. a Analysis of IL-15Rβ and γc expression on C2C12 myoblasts under the same condition of Fig. 2a. Data are representative of three independent experiments with similar results. b Expression of Il15rb and γc mRNA in C2C12 and primary myotube. Samples were collected 24 h after cytokine treatment and analyzed by qPCR. Data are triplicates and representative of two independent experiments with similar results. c Immunoblotting of STAT5 phosphorylation in C2C12 myotubes after treating with IL-15 or IL-15 hyperagonist for 30 min. Quantification data of four independent experiments are shown below. d Anti-IL-15Rβ antibody diminished IL-15 hyperagonist-induced STAT5 phosphorylation in C2C12 myotubes. Myotubes were pretreated with anti-IL-15Rβ or isotype control antibody for 1 h and then treated with IL-15 hyperagonist for 30 min. Data are representative of two independent experiments with similar results. e Immunoblotting of STAT5 phosphorylation in C2C12 myotubes that were first stimulated with TNF-α and IFN-γ for 24 h and then treated with exogenous IL-15 or IL-15 hyperagonist for 30 min. Data are representative of two independent experiments with similar results. f Accumulation of myosin heavy chain (MyHC) in C2C12 myotubes treated with IL-15 (50 and 100 ng/ml), IL-15 hyperagonist (50 and 100 ng/ml), or vehicle (0 ng/ml). After 48 h, cells were immunostained with FITC-labeled anti-myosin antibody to evaluate the accumulation of MyHC. MyHC-positive area per microscopic field are shown in the right panel and represented as the percentages of con. Quantification data were pooled from three independent experiments. Each symbol represents the quantification data from one microscopic field. Scale bar = 500 μm. Data are mean ± SEM. *p < 0.05, **p < 0.01, ***p < 0.001

Mentions: Despite of various reported IL-15 functions in the skeletal muscle [12–16], the IL-15-induced signals in skeletal muscle cells remains unexplored. We first examined the expression of IL-15Rβ and γc on C2C12 myoblasts by flow cytometry and detected no IL-15Rβ expression but a low level of γc induced by TNF-α and IFN-γ treatment (Fig. 3a). The more sensitive qPCR also detected induction of γc mRNA, but not Il15rb mRNA, in C2C12 and primary myotubes by the cytokines (Fig. 3b). Moreover, the level of Il15rb mRNA in the primary myotube was 16 times lower than that in the C2C12 myotube based on the Ct value of qPCR.Fig. 3


Skeletal muscle interleukin 15 promotes CD8(+) T-cell function and autoimmune myositis.

Huang PL, Hou MS, Wang SW, Chang CL, Liou YH, Liao NS - Skelet Muscle (2015)

High concentration of IL-15 hyperagonist, but not IL-15, induces STAT5 signaling and atrophy in skeletal muscle cells. a Analysis of IL-15Rβ and γc expression on C2C12 myoblasts under the same condition of Fig. 2a. Data are representative of three independent experiments with similar results. b Expression of Il15rb and γc mRNA in C2C12 and primary myotube. Samples were collected 24 h after cytokine treatment and analyzed by qPCR. Data are triplicates and representative of two independent experiments with similar results. c Immunoblotting of STAT5 phosphorylation in C2C12 myotubes after treating with IL-15 or IL-15 hyperagonist for 30 min. Quantification data of four independent experiments are shown below. d Anti-IL-15Rβ antibody diminished IL-15 hyperagonist-induced STAT5 phosphorylation in C2C12 myotubes. Myotubes were pretreated with anti-IL-15Rβ or isotype control antibody for 1 h and then treated with IL-15 hyperagonist for 30 min. Data are representative of two independent experiments with similar results. e Immunoblotting of STAT5 phosphorylation in C2C12 myotubes that were first stimulated with TNF-α and IFN-γ for 24 h and then treated with exogenous IL-15 or IL-15 hyperagonist for 30 min. Data are representative of two independent experiments with similar results. f Accumulation of myosin heavy chain (MyHC) in C2C12 myotubes treated with IL-15 (50 and 100 ng/ml), IL-15 hyperagonist (50 and 100 ng/ml), or vehicle (0 ng/ml). After 48 h, cells were immunostained with FITC-labeled anti-myosin antibody to evaluate the accumulation of MyHC. MyHC-positive area per microscopic field are shown in the right panel and represented as the percentages of con. Quantification data were pooled from three independent experiments. Each symbol represents the quantification data from one microscopic field. Scale bar = 500 μm. Data are mean ± SEM. *p < 0.05, **p < 0.01, ***p < 0.001
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Related In: Results  -  Collection

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Fig3: High concentration of IL-15 hyperagonist, but not IL-15, induces STAT5 signaling and atrophy in skeletal muscle cells. a Analysis of IL-15Rβ and γc expression on C2C12 myoblasts under the same condition of Fig. 2a. Data are representative of three independent experiments with similar results. b Expression of Il15rb and γc mRNA in C2C12 and primary myotube. Samples were collected 24 h after cytokine treatment and analyzed by qPCR. Data are triplicates and representative of two independent experiments with similar results. c Immunoblotting of STAT5 phosphorylation in C2C12 myotubes after treating with IL-15 or IL-15 hyperagonist for 30 min. Quantification data of four independent experiments are shown below. d Anti-IL-15Rβ antibody diminished IL-15 hyperagonist-induced STAT5 phosphorylation in C2C12 myotubes. Myotubes were pretreated with anti-IL-15Rβ or isotype control antibody for 1 h and then treated with IL-15 hyperagonist for 30 min. Data are representative of two independent experiments with similar results. e Immunoblotting of STAT5 phosphorylation in C2C12 myotubes that were first stimulated with TNF-α and IFN-γ for 24 h and then treated with exogenous IL-15 or IL-15 hyperagonist for 30 min. Data are representative of two independent experiments with similar results. f Accumulation of myosin heavy chain (MyHC) in C2C12 myotubes treated with IL-15 (50 and 100 ng/ml), IL-15 hyperagonist (50 and 100 ng/ml), or vehicle (0 ng/ml). After 48 h, cells were immunostained with FITC-labeled anti-myosin antibody to evaluate the accumulation of MyHC. MyHC-positive area per microscopic field are shown in the right panel and represented as the percentages of con. Quantification data were pooled from three independent experiments. Each symbol represents the quantification data from one microscopic field. Scale bar = 500 μm. Data are mean ± SEM. *p < 0.05, **p < 0.01, ***p < 0.001
Mentions: Despite of various reported IL-15 functions in the skeletal muscle [12–16], the IL-15-induced signals in skeletal muscle cells remains unexplored. We first examined the expression of IL-15Rβ and γc on C2C12 myoblasts by flow cytometry and detected no IL-15Rβ expression but a low level of γc induced by TNF-α and IFN-γ treatment (Fig. 3a). The more sensitive qPCR also detected induction of γc mRNA, but not Il15rb mRNA, in C2C12 and primary myotubes by the cytokines (Fig. 3b). Moreover, the level of Il15rb mRNA in the primary myotube was 16 times lower than that in the C2C12 myotube based on the Ct value of qPCR.Fig. 3

Bottom Line: 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.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.

View Article: PubMed Central - PubMed

Affiliation: Molecular Cell Biology, Taiwan International Graduate Program, Institute of Molecular Biology, Academia Sinica, and Graduate Institute of Life Sciences, National Defense Medical Center, Taipei, Taiwan ; Institute of Molecular Biology, Academia Sinica, Taipei, 11529 Taiwan.

ABSTRACT

Background: 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.

Methods: 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.

Results: 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.

Conclusions: 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.

No MeSH data available.


Related in: MedlinePlus