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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

Skeletal muscle cells express IL-15/IL-15Rα protein complex in response to TNF-α and IFN-γ stimulation. a Expression of Il15 and Il15ra mRNA during C2C12 myoblast differentiation. Samples were collected before (0) and 2, 4, and 6 days after differentiation induction. b Expression of Il15 and Il15ra mRNA in C2C12 myotubes treated with TNF-α (10 ng/ml), IFN-γ (10 ng/ml), TNF-α + IFN-γ (TNF + IFN, 10 ng/ml each), or without cytokine (con) for 1, 2, 3, 6, 12, and 24 h. c Expression of Il15 and Il15ra mRNA in primary myotubes treated with TNF-α (5 ng/ml), IFN-γ (5 ng/ml), TNF + IFN (5 ng/ml each), or without cytokine (con) for 24 h. d Expression of IL-15/IL-15Rα complex protein in C2C12 myoblasts and myotubes treated with TNF-α (1 or 10 ng/ml), IFN-γ (1 or 10 ng/ml), TNF + IFN (1 or 10 ng/ml each), or without cytokine (con) for 24 h. e Expression of IL-15/IL-15Rα complex protein in primary myotubes treated with TNF + IFN (5 ng/ml each) or vehicle (con) for 24 h. f Expression of IL-15/IL-15Rα complex protein in skeletal muscle in vivo. TNF-α plus IFN-γ (1 mg each/injection) or PBS (con) was injected into the quadriceps muscles of mice three times at 4-h intervals. The injected muscles were collected 16 h after the last injection. Total RNA was isolated and analyzed by qPCR (a–c). Protein lysate and cell culture medium were collected and measured by ELISA (d–f). Data in (a–c) were triplicates and representative of two independent experiments with similar result. Data in (d, e) were pooled from three independent experiments. Data in (f) was pooled from three mice in each group. Data are mean ± SEM. *p < 0.05, **p < 0.01, ***p < 0.001, in comparison to “0” or “con”
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Fig1: Skeletal muscle cells express IL-15/IL-15Rα protein complex in response to TNF-α and IFN-γ stimulation. a Expression of Il15 and Il15ra mRNA during C2C12 myoblast differentiation. Samples were collected before (0) and 2, 4, and 6 days after differentiation induction. b Expression of Il15 and Il15ra mRNA in C2C12 myotubes treated with TNF-α (10 ng/ml), IFN-γ (10 ng/ml), TNF-α + IFN-γ (TNF + IFN, 10 ng/ml each), or without cytokine (con) for 1, 2, 3, 6, 12, and 24 h. c Expression of Il15 and Il15ra mRNA in primary myotubes treated with TNF-α (5 ng/ml), IFN-γ (5 ng/ml), TNF + IFN (5 ng/ml each), or without cytokine (con) for 24 h. d Expression of IL-15/IL-15Rα complex protein in C2C12 myoblasts and myotubes treated with TNF-α (1 or 10 ng/ml), IFN-γ (1 or 10 ng/ml), TNF + IFN (1 or 10 ng/ml each), or without cytokine (con) for 24 h. e Expression of IL-15/IL-15Rα complex protein in primary myotubes treated with TNF + IFN (5 ng/ml each) or vehicle (con) for 24 h. f Expression of IL-15/IL-15Rα complex protein in skeletal muscle in vivo. TNF-α plus IFN-γ (1 mg each/injection) or PBS (con) was injected into the quadriceps muscles of mice three times at 4-h intervals. The injected muscles were collected 16 h after the last injection. Total RNA was isolated and analyzed by qPCR (a–c). Protein lysate and cell culture medium were collected and measured by ELISA (d–f). Data in (a–c) were triplicates and representative of two independent experiments with similar result. Data in (d, e) were pooled from three independent experiments. Data in (f) was pooled from three mice in each group. Data are mean ± SEM. *p < 0.05, **p < 0.01, ***p < 0.001, in comparison to “0” or “con”

Mentions: C2C12 myoblasts were maintained in Dulbecco’s modified Eagle’s medium (DMEM) containing 10 % fetal bovine serum (FBS). Confluent C2C12 myoblasts were shifted to differentiation medium (DMEM containing 2 % horse serum) for myotube differentiation. Unless indicated otherwise (Fig. 1a), C2C12 myotubes were used 4 days after differentiation induction, when about 80 % of culture plate surface was covered by myotubes. Primary myoblasts were isolated from the limb muscle of 1- to 3-day-old neonatal mice and purified by sorting of α7 integrin-positive cells as previously described [34]. Rat anti-α7 integrin monoclonal antibody, CA5.5, was kindly provided by Dr. Chung-Chen Yao (National Taiwan University). Purified primary myoblasts (about 25,000 cells/cm2) were cultured in growth medium (40 % Ham’s F-10, 40 % DMEM, 20 % FBS, 2.5 ng/ml bFGF) for 1 day and then switched to differentiation medium (DMEM containing 5 % horse serum). Some primary myoblasts already fused to form nascent myotubes during the 1-day culture in growth medium. After changing to differentiation medium, well-differentiated primary myotubes appeared in day 1 and were used for experiments in day 2.Fig. 1


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)

Skeletal muscle cells express IL-15/IL-15Rα protein complex in response to TNF-α and IFN-γ stimulation. a Expression of Il15 and Il15ra mRNA during C2C12 myoblast differentiation. Samples were collected before (0) and 2, 4, and 6 days after differentiation induction. b Expression of Il15 and Il15ra mRNA in C2C12 myotubes treated with TNF-α (10 ng/ml), IFN-γ (10 ng/ml), TNF-α + IFN-γ (TNF + IFN, 10 ng/ml each), or without cytokine (con) for 1, 2, 3, 6, 12, and 24 h. c Expression of Il15 and Il15ra mRNA in primary myotubes treated with TNF-α (5 ng/ml), IFN-γ (5 ng/ml), TNF + IFN (5 ng/ml each), or without cytokine (con) for 24 h. d Expression of IL-15/IL-15Rα complex protein in C2C12 myoblasts and myotubes treated with TNF-α (1 or 10 ng/ml), IFN-γ (1 or 10 ng/ml), TNF + IFN (1 or 10 ng/ml each), or without cytokine (con) for 24 h. e Expression of IL-15/IL-15Rα complex protein in primary myotubes treated with TNF + IFN (5 ng/ml each) or vehicle (con) for 24 h. f Expression of IL-15/IL-15Rα complex protein in skeletal muscle in vivo. TNF-α plus IFN-γ (1 mg each/injection) or PBS (con) was injected into the quadriceps muscles of mice three times at 4-h intervals. The injected muscles were collected 16 h after the last injection. Total RNA was isolated and analyzed by qPCR (a–c). Protein lysate and cell culture medium were collected and measured by ELISA (d–f). Data in (a–c) were triplicates and representative of two independent experiments with similar result. Data in (d, e) were pooled from three independent experiments. Data in (f) was pooled from three mice in each group. Data are mean ± SEM. *p < 0.05, **p < 0.01, ***p < 0.001, in comparison to “0” or “con”
© Copyright Policy - OpenAccess
Related In: Results  -  Collection

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getmorefigures.php?uid=PMC4584479&req=5

Fig1: Skeletal muscle cells express IL-15/IL-15Rα protein complex in response to TNF-α and IFN-γ stimulation. a Expression of Il15 and Il15ra mRNA during C2C12 myoblast differentiation. Samples were collected before (0) and 2, 4, and 6 days after differentiation induction. b Expression of Il15 and Il15ra mRNA in C2C12 myotubes treated with TNF-α (10 ng/ml), IFN-γ (10 ng/ml), TNF-α + IFN-γ (TNF + IFN, 10 ng/ml each), or without cytokine (con) for 1, 2, 3, 6, 12, and 24 h. c Expression of Il15 and Il15ra mRNA in primary myotubes treated with TNF-α (5 ng/ml), IFN-γ (5 ng/ml), TNF + IFN (5 ng/ml each), or without cytokine (con) for 24 h. d Expression of IL-15/IL-15Rα complex protein in C2C12 myoblasts and myotubes treated with TNF-α (1 or 10 ng/ml), IFN-γ (1 or 10 ng/ml), TNF + IFN (1 or 10 ng/ml each), or without cytokine (con) for 24 h. e Expression of IL-15/IL-15Rα complex protein in primary myotubes treated with TNF + IFN (5 ng/ml each) or vehicle (con) for 24 h. f Expression of IL-15/IL-15Rα complex protein in skeletal muscle in vivo. TNF-α plus IFN-γ (1 mg each/injection) or PBS (con) was injected into the quadriceps muscles of mice three times at 4-h intervals. The injected muscles were collected 16 h after the last injection. Total RNA was isolated and analyzed by qPCR (a–c). Protein lysate and cell culture medium were collected and measured by ELISA (d–f). Data in (a–c) were triplicates and representative of two independent experiments with similar result. Data in (d, e) were pooled from three independent experiments. Data in (f) was pooled from three mice in each group. Data are mean ± SEM. *p < 0.05, **p < 0.01, ***p < 0.001, in comparison to “0” or “con”
Mentions: C2C12 myoblasts were maintained in Dulbecco’s modified Eagle’s medium (DMEM) containing 10 % fetal bovine serum (FBS). Confluent C2C12 myoblasts were shifted to differentiation medium (DMEM containing 2 % horse serum) for myotube differentiation. Unless indicated otherwise (Fig. 1a), C2C12 myotubes were used 4 days after differentiation induction, when about 80 % of culture plate surface was covered by myotubes. Primary myoblasts were isolated from the limb muscle of 1- to 3-day-old neonatal mice and purified by sorting of α7 integrin-positive cells as previously described [34]. Rat anti-α7 integrin monoclonal antibody, CA5.5, was kindly provided by Dr. Chung-Chen Yao (National Taiwan University). Purified primary myoblasts (about 25,000 cells/cm2) were cultured in growth medium (40 % Ham’s F-10, 40 % DMEM, 20 % FBS, 2.5 ng/ml bFGF) for 1 day and then switched to differentiation medium (DMEM containing 5 % horse serum). Some primary myoblasts already fused to form nascent myotubes during the 1-day culture in growth medium. After changing to differentiation medium, well-differentiated primary myotubes appeared in day 1 and were used for experiments in day 2.Fig. 1

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