Human Muscle Progenitor Cells Displayed Immunosuppressive Effect through Galectin-1 and Semaphorin-3A

Abstract

In human skeletal muscle, myoblasts represent the main population of myogenic progenitors. We previously showed that, beside their myogenic differentiation capacities, myoblasts also differentiate towards osteogenic and chondrogenic lineages, some properties generally considered being hallmarks of mesenchymal stem cells (MSCs). MSCs are also characterized by their immunosuppressive potential, through cell-cell contacts and soluble factors, including prostaglandin E-2 (PGE-2), transforming growth factor-β1 (TGF-β1), interleukine-10, or indoleamine 2,3-dioxygenase. We and others also reported that Galectin-1 (Gal-1) and Semaphorin-3A (Sema-3A) were involved in MSCs-mediated immunosuppression. Here, we show that human myoblasts induce a significant and dose-dependant proliferation inhibition, independently of PGE-2 and TGF-β1. Our experiments revealed that myoblasts, in culture or in situ in human muscles, expressed and secreted Gal-1 and Sema-3A. Furthermore, myoblasts immunosuppressive functions were reverted by using blocking antibodies against Gal-1 or Sema-3A. Together, these results demonstrate an unsuspected immunosuppressive effect of myoblasts that may open new therapeutic perspectives.

Figure 1

Human myoblasts characterization and PBMCs proliferation inhibition. (a) Immunophenotyping by flow cytometry analysis: cells were labeled with specific antibodies (red line) or isotype control (green line). (b) Myogenic differentiation ability of CD56+ cells. Myotubes expressed myosin heavy chain (red), nuclei were labeled with DAPI (blue). (c) PBMCs were cultured in the presence of increasing number of myoblasts at ratio ranging from 1/1000 to 1/5. Data are means ± SEM of triplicates from one representative of at least 10 independent experiments. P values from Student's t-test are indicated (**P < 0.01, ***P < 0.001).

Figure 2

Human myoblasts inhibit PBMCs proliferation independently of PGE-2 and TGFβ-1. (a) PGE-2 (left panel) and TGF-β (right panel) levels were assessed by ELISA on culture supernatants of MSCs and myoblasts, in the presence (black bars) or absence (white bars) of PBMCs. (b) TGF-β1 blocking antibodies (10 to 40 μg/mL) were used to neutralize the immunosuppressive activity of myoblasts on PBMCs. PBMCs and myoblasts were cultured at a ratio 1/5.

Figure 3

Human myoblasts immunosuppression is mediated by Gal-1 and Sema-3A. (a, b, c) RT-PCR, flow cytometric, and immunoprecipitation analysis of Gal-1 and Sema-3A expression and secretion by myoblasts. MSCs were used as control. Data are means ± SEM of triplicates from one representative of at least 10 independent experiments. P values from Student's t-test are indicated (**P < 0.01, ***P < 0.001). (d) Neutralization of Gal-1 and Sema-3A restores PBMCs proliferation. PBMCs from eight independent donors were cultured alone (A+B) or with myoblasts (ratio 1/5) in the presence of increasing concentration of anti-Gal-1 or anti-Sema-3A antibodies (0 to 40 μg/mL). Data are means ± SD of triplicates from one representative of at least 10 independent experiments. P values from Student's t-test are indicated (*P <0.05, **P < 0.01, and ***P < 0.001).

Figure 4

In situ Gal-1 and Sema-3A expression. (a) Microscopy analysis of Gal-1 (green) expressions in situ in normal human muscle. Bar represents 100 μm. (b) Microscopy analysis of Sema-3A (red) expressions in situ in normal human muscle. Bar represents 100 μm. Nuclei were stained with Dapi and appeared in blue.