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. 2018 May 1;9(5):551.
doi: 10.1038/s41419-018-0426-z.

Activated dendritic cells modulate proliferation and differentiation of human myoblasts

Leandro Ladislau  1   2 , Débora M Portilho  2 , Tristan Courau  3 , Alhondra Solares-Pérez  2 , Elisa Negroni  2 , Jeanne Lainé  2 , David Klatzmann  3 , Adriana Bonomo  4 , Yves Allenbach  2 , Olivier Benveniste  2 , Ingo Riederer  4   5 , Wilson Savino  4   5 , Vincent Mouly  2 , Gillian Butler-Browne  2 , Claudia F Benjamim  6
Affiliations

Activated dendritic cells modulate proliferation and differentiation of human myoblasts

Leandro Ladislau et al. Cell Death Dis. .

Erratum in

Abstract

Idiopathic Inflammatory Myopathies (IIMs) are a heterogeneous group of autoimmune diseases affecting skeletal muscle tissue homeostasis. They are characterized by muscle weakness and inflammatory infiltration with tissue damage. Amongst the cells in the muscle inflammatory infiltration, dendritic cells (DCs) are potent antigen-presenting and key components in autoimmunity exhibiting an increased activation in inflamed tissues. Since, the IIMs are characterized by the focal necrosis/regeneration and muscle atrophy, we hypothesized that DCs may play a role in these processes. Due to the absence of a reliable in vivo model for IIMs, we first performed co-culture experiments with immature DCs (iDC) or LPS-activated DCs (actDC) and proliferating myoblasts or differentiating myotubes. We demonstrated that both iDC or actDCs tightly interact with myoblasts and myotubes, increased myoblast proliferation and migration, but inhibited myotube differentiation. We also observed that actDCs increased HLA-ABC, HLA-DR, VLA-5, and VLA-6 expression and induced cytokine secretion on myoblasts. In an in vivo regeneration model, the co-injection of human myoblasts and DCs enhanced human myoblast migration, whereas the absolute number of human myofibres was unchanged. In conclusion, we suggest that in the early stages of myositis, DCs may play a crucial role in inducing muscle-damage through cell-cell contact and inflammatory cytokine secretion, leading to muscle regeneration impairment.

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Conflict of interest statement

The authors declare that they have no conflict of interest.

Figures

Fig. 1
Fig. 1. Close contact during co-culture between DCs and myoblasts or myotubes.
A Phase contrast microscopy of myoblasts co-cultured with actDCs (LPS 100 ng/mL) (a and b) for 48 h. a represents a low magnification and b a higher magnification. Bars represent 100 μm. B Transmission electron microscopy of myoblasts and actDCs (LPS 100 ng/mL) co-cultured in proliferation medium for 48 h (a). Different magnifications of the close contact area of myoblast (MB) and DC1 (b, c, d), while (d) is the high magnification of the rectangle (h) in (c). Different magnifications of the close contact area of myoblast (MB) and DC2 (e, f, g), while (f, g) are high magnifications of the rectangles (i, j) seen in (e), respectively. Bars represent 5, 2, 1, 100, and 500 nm. Data are representative of three independent experiments
Fig. 2
Fig. 2. Confocal images of close contact between actDCs and myoblasts.
Myoblasts were co-cultured with actDCs for 48 h, fixed and stained for image acquisition. Two different fields were captured to evaluate the close contact between the cells. a and b represent the Z-axis stacks of one field, whereas c and d represent the other field. Red desmin, green CD11b, blue nucleus, purple cadherin. The white arrow indicates the putative cell contact. The white square highlights the engulfment of the DCs by the myoblast. Data are representative of two different experiments
Fig. 3
Fig. 3. HLA-ABC and HLA-DR expression on myoblasts is increased during the co-culture with actDCs.
Myoblasts were seeded in co-culture with iDC or actDCs, the cells were recovered, stained and analyzed by flow cytometry. a Dot-plots from myoblasts, myoblasts incubated with LPS (100 ng/mL), myoblasts incubated with iDC and myoblasts incubated with actDCs. The gated cells are CD56+ myoblasts. b, c reveal HLA-ABC and HLA-DR histograms, respectively, from myoblasts and DC co-culture, discriminated as black line myoblasts, gray line myoblasts + LPS, red line myoblasts + iDC, blue line myoblasts + actDCs. Data are representative of three different experiments
Fig. 4
Fig. 4. DCs incubation increase the proliferation and migration of myoblasts.
a Myoblasts were co-cultured with iDC or actDCs for 48 h and the proliferation was evaluated by BrdU incorporation. b Quantitative analysis of the proliferation assay. The data are representative of three experiments. c Images representative of scratched areas from myoblasts alone or after incubation with LPS, or co-cultured with iDC or actDCs for 48 h. d The data are expressed as the percentage of the closed area. Data shown as means ± SE of duplicate wells and are representative of three different experiments. Bars represent 100 μm. *p < 0.05; ***p < 0.001, and ****p < 0.0001 compared to myoblasts
Fig. 5
Fig. 5. DCs inhibit myotube differentiation.
a Immunostaining for myogenin (red), myosin heavy chain (green), and nuclei (Hoechst-blue) of myoblasts, myoblasts stimulated with LPS or co-cultured with iDC or actDCs after 24 and 96 h of incubation. The data represent three independent experiments. Bars represent 100 μm. b–d Graph representative of the fusion index, myogenin and MyoD expression presented in panel A evaluated at 24 to 96 h after incubation. e–f Fusion index of two different adult myoblast co-culture obtained from a 25 (e) and a 60-year-old (f) healthy donors evaluated at 96 h after incubation. Data shown as means ± SE of duplicates and are representative of three different experiments. *p < 0.05, **p < 0.01, and ***p < 0.001 compared to myoblasts
Fig. 6
Fig. 6. Co-injection of human myoblasts and DCs into the Tibialis anterior (TA) muscle pre-injured by cryolesion in Rag-/-IL2R-/- mice.
a Immunostaining for human specific lamin A/C (magenta) and spectrin (green) on muscle sections obtained 30 days after cryolesion of the tibialis anterior of Rag−/−IL2R−/− mice. The injections of myoblasts, myoblasts plus iDC or myoblasts plus actDCs were made in 3–4 TA muscles per group. Bars represent 100 μm. The graphs show the number of human nuclei (b), human spectrin postive muscle fibers (c), and cell dispersion (d). The data represent one experiment. Myoblasts were seeded in co-culture with iDC or actDCs, and were recovered, stained and analyzed by flow cytometry. CD49e (e) and CD49f (f) histograms from myoblasts and DC co-cultures, discriminated as black line myoblasts, gray line myoblasts plus LPS, red line myoblasts plus iDC, and blue line myoblasts plus actDCs. Data are representative of 4 different TA muscles
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