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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 presented antigen and provided IL-15 to promote the effector function of CD8+ T cells under TNF-α and IFN-γ treatment. a Expression of IL-15 and H-2Kb on parental C2C12 and C2C12-Kb/OVA cells. Cells were treated with TNF-α and IFN-γ (TNF + IFN) or without (Control) for 24 h, stained with anti-IL-15 or anti-H-2Kb antibody, then analyzed by flow cytometry. “Unstained” indicates C2C12 or C2C12-Kb/OVA myoblast without antibody staining. b Expression of grB and IFN-γ by CD8+ T cells co-cultured with cytokine-treated C2C12-Kb or C2C12-Kb/OVA myoblasts for 8 h. After co-culture, CD8+ T cells were stained with anti-grB and IFN-γ antibody by intracellular staining then analyzed by flow cytometry. Data are representative of three independent experiments with similar results. c Effect of anti-IL-2 (αIL-2, S4B6) or anti-IL-2/15Rβ (αRβ, TM-β1) antibody on grB and IFN-γ production by CD8+ T cells co-cultured with TNF + IFN-pretreated C2C12-Kb/OVA myoblasts. The percentages of grB+ and IFN-γ+ cells in CD8+ T cells are indicated in the dot plots. Data from independent experiments were compiled as % normalized activation using the percentage of grB+ or IFN-γ+ cells in CD8+ T cells of the TNF + IFN-pretreated C2C12-Kb/OVA cell co-culture group of each experiment as 100 % (bottom panels). Isotype controls, Rat IgG2a (Iso) and Rat IgG2b (Iso). Each symbol is representative of one independent experiment. **p < 0.01, ***p < 0.001
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Fig5: Skeletal muscle cells presented antigen and provided IL-15 to promote the effector function of CD8+ T cells under TNF-α and IFN-γ treatment. a Expression of IL-15 and H-2Kb on parental C2C12 and C2C12-Kb/OVA cells. Cells were treated with TNF-α and IFN-γ (TNF + IFN) or without (Control) for 24 h, stained with anti-IL-15 or anti-H-2Kb antibody, then analyzed by flow cytometry. “Unstained” indicates C2C12 or C2C12-Kb/OVA myoblast without antibody staining. b Expression of grB and IFN-γ by CD8+ T cells co-cultured with cytokine-treated C2C12-Kb or C2C12-Kb/OVA myoblasts for 8 h. After co-culture, CD8+ T cells were stained with anti-grB and IFN-γ antibody by intracellular staining then analyzed by flow cytometry. Data are representative of three independent experiments with similar results. c Effect of anti-IL-2 (αIL-2, S4B6) or anti-IL-2/15Rβ (αRβ, TM-β1) antibody on grB and IFN-γ production by CD8+ T cells co-cultured with TNF + IFN-pretreated C2C12-Kb/OVA myoblasts. The percentages of grB+ and IFN-γ+ cells in CD8+ T cells are indicated in the dot plots. Data from independent experiments were compiled as % normalized activation using the percentage of grB+ or IFN-γ+ cells in CD8+ T cells of the TNF + IFN-pretreated C2C12-Kb/OVA cell co-culture group of each experiment as 100 % (bottom panels). Isotype controls, Rat IgG2a (Iso) and Rat IgG2b (Iso). Each symbol is representative of one independent experiment. **p < 0.01, ***p < 0.001

Mentions: IL-15 is a well-known survival and activation factor for memory CD8+ T cells. As inflammatory cytokines induce the expression of IL-15/IL-15Rα and antigen presentation molecules by myoblasts and myotubes [49], we designed a muscle-cell-T-cell co-culture system to assess whether the muscle cells directly activate CD8+ T cells and the role of IL-15 in this process. We generated a C2C12 myoblast subline that stably expresses H-2Kb-EGFP (C2C12-Kb). As overexpression of H-2Kb impairs myoblast differentiation in this study and [50], this co-culture system is for myoblast and T cells. We then transduced full-length OVA gene into C2C12-Kb myoblast as an endogenous antigen and generated C2C12-Kb/OVA myoblast. TNF-α and IFN-γ greatly induced the expression of IL-15 and H-2Kb on C2C12-Kb/OVA cells (Fig. 5a). This high induction of H-2Kb might partly result from the up-regulation of β2-microglobulin (B2m) by the cytokines (Fig. 4b), because B2m is essential for the stabilization of MHC class I molecule in correct conformation to receive the peptide in the ER and to move from the ER to the cell surface [51]. The cytokine-stimulated C2C12-Kb/OVA, but not C2C12-Kb, cells induced production of granzyme B (grB) and IFN-γ by memory-like OT-1 cells (Fig. 5b). We then found that an IL-15Rβ-blocking antibody, but not IL-2-neutralizing antibody, suppressed grB and IFN-γ production by the memory-like OT-1 cells (Fig. 5c). As the cytokine-stimulated C2C12-Kb/OVA cells were washed before co-culturing with OT-1 cells in the presence of the exocytosis inhibitor brefeldin A, IL-15 was presumably only present on the muscle cell surface. These results indicate that myoblasts stimulated with TNF-α and IFN-γ present antigen and IL-15 to memory-like CD8+ T cells to promote their effector function. Given that both myoblasts and myotubes function as antigen-presenting cells under inflammation, what we observed in the myoblast-CD8+ T-cell co-culture is likely applicable to myotubes.Fig. 5


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 presented antigen and provided IL-15 to promote the effector function of CD8+ T cells under TNF-α and IFN-γ treatment. a Expression of IL-15 and H-2Kb on parental C2C12 and C2C12-Kb/OVA cells. Cells were treated with TNF-α and IFN-γ (TNF + IFN) or without (Control) for 24 h, stained with anti-IL-15 or anti-H-2Kb antibody, then analyzed by flow cytometry. “Unstained” indicates C2C12 or C2C12-Kb/OVA myoblast without antibody staining. b Expression of grB and IFN-γ by CD8+ T cells co-cultured with cytokine-treated C2C12-Kb or C2C12-Kb/OVA myoblasts for 8 h. After co-culture, CD8+ T cells were stained with anti-grB and IFN-γ antibody by intracellular staining then analyzed by flow cytometry. Data are representative of three independent experiments with similar results. c Effect of anti-IL-2 (αIL-2, S4B6) or anti-IL-2/15Rβ (αRβ, TM-β1) antibody on grB and IFN-γ production by CD8+ T cells co-cultured with TNF + IFN-pretreated C2C12-Kb/OVA myoblasts. The percentages of grB+ and IFN-γ+ cells in CD8+ T cells are indicated in the dot plots. Data from independent experiments were compiled as % normalized activation using the percentage of grB+ or IFN-γ+ cells in CD8+ T cells of the TNF + IFN-pretreated C2C12-Kb/OVA cell co-culture group of each experiment as 100 % (bottom panels). Isotype controls, Rat IgG2a (Iso) and Rat IgG2b (Iso). Each symbol is representative of one independent experiment. **p < 0.01, ***p < 0.001
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Fig5: Skeletal muscle cells presented antigen and provided IL-15 to promote the effector function of CD8+ T cells under TNF-α and IFN-γ treatment. a Expression of IL-15 and H-2Kb on parental C2C12 and C2C12-Kb/OVA cells. Cells were treated with TNF-α and IFN-γ (TNF + IFN) or without (Control) for 24 h, stained with anti-IL-15 or anti-H-2Kb antibody, then analyzed by flow cytometry. “Unstained” indicates C2C12 or C2C12-Kb/OVA myoblast without antibody staining. b Expression of grB and IFN-γ by CD8+ T cells co-cultured with cytokine-treated C2C12-Kb or C2C12-Kb/OVA myoblasts for 8 h. After co-culture, CD8+ T cells were stained with anti-grB and IFN-γ antibody by intracellular staining then analyzed by flow cytometry. Data are representative of three independent experiments with similar results. c Effect of anti-IL-2 (αIL-2, S4B6) or anti-IL-2/15Rβ (αRβ, TM-β1) antibody on grB and IFN-γ production by CD8+ T cells co-cultured with TNF + IFN-pretreated C2C12-Kb/OVA myoblasts. The percentages of grB+ and IFN-γ+ cells in CD8+ T cells are indicated in the dot plots. Data from independent experiments were compiled as % normalized activation using the percentage of grB+ or IFN-γ+ cells in CD8+ T cells of the TNF + IFN-pretreated C2C12-Kb/OVA cell co-culture group of each experiment as 100 % (bottom panels). Isotype controls, Rat IgG2a (Iso) and Rat IgG2b (Iso). Each symbol is representative of one independent experiment. **p < 0.01, ***p < 0.001
Mentions: IL-15 is a well-known survival and activation factor for memory CD8+ T cells. As inflammatory cytokines induce the expression of IL-15/IL-15Rα and antigen presentation molecules by myoblasts and myotubes [49], we designed a muscle-cell-T-cell co-culture system to assess whether the muscle cells directly activate CD8+ T cells and the role of IL-15 in this process. We generated a C2C12 myoblast subline that stably expresses H-2Kb-EGFP (C2C12-Kb). As overexpression of H-2Kb impairs myoblast differentiation in this study and [50], this co-culture system is for myoblast and T cells. We then transduced full-length OVA gene into C2C12-Kb myoblast as an endogenous antigen and generated C2C12-Kb/OVA myoblast. TNF-α and IFN-γ greatly induced the expression of IL-15 and H-2Kb on C2C12-Kb/OVA cells (Fig. 5a). This high induction of H-2Kb might partly result from the up-regulation of β2-microglobulin (B2m) by the cytokines (Fig. 4b), because B2m is essential for the stabilization of MHC class I molecule in correct conformation to receive the peptide in the ER and to move from the ER to the cell surface [51]. The cytokine-stimulated C2C12-Kb/OVA, but not C2C12-Kb, cells induced production of granzyme B (grB) and IFN-γ by memory-like OT-1 cells (Fig. 5b). We then found that an IL-15Rβ-blocking antibody, but not IL-2-neutralizing antibody, suppressed grB and IFN-γ production by the memory-like OT-1 cells (Fig. 5c). As the cytokine-stimulated C2C12-Kb/OVA cells were washed before co-culturing with OT-1 cells in the presence of the exocytosis inhibitor brefeldin A, IL-15 was presumably only present on the muscle cell surface. These results indicate that myoblasts stimulated with TNF-α and IFN-γ present antigen and IL-15 to memory-like CD8+ T cells to promote their effector function. Given that both myoblasts and myotubes function as antigen-presenting cells under inflammation, what we observed in the myoblast-CD8+ T-cell co-culture is likely applicable to myotubes.Fig. 5

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