De Bleecker, Jan

Gonçalves, Amanda

Herbelet, Sandrine

Vandesompele, Jo

Ghent University Academic Bibliography

The proliferation of Duchenne muscular dystrophy fibroblasts cultured under inflammatory conditions is reduced by methylprednisolone through  modulation of NFAT5 localization in the cell  Sandrine Herbelet1,*, Amanda Gonçalves2, Jo Vandesompele3, Jan L. De Bleecker1,*    Affiliations and references 1:  Department of Neurology, Ghent University and Ghent University Hospital, Ghent, Belgium                                                                                                                                                                 2: 1. VIB Inflammation Research Center, Ghent, Belgium; 2. Department of Biomedical Molecular Biology, Ghent University, Ghent, Belgium; 3. VIB Bio          Imaging Core Gent, Ghent, Belgium                                                                                                                                   3: Center for Medical Genetics and Cancer Research Institute Ghent (CRIG), Ghent, Belgium   References 1) Abdel-Salam, E., Abdel-Mequi, I., Korraa, S.S. (2009). Markers of degeneration and regeneration in Duchenne muscular dystrophy. Acta Myol. 28, 94-100. 2) Banker, B.Q. and Engel, A.G. (2004) Basic reactions of muscle. In: Engel AG, Franzini-Armstrong C (eds) Myology. McGraw-Hill, New York, pp 691–748. 3) Kendall, R.T. and Feghali-Bostwick, C.A. (2014). Fibroblasts in fibrosis: novel roles and mediators. Front. Pharmacol. 5, article 123, doi: 10.3389/fphar.2014.00123 4) Mann , C.J., Perdiguero, E., Kharraz, Y., Aguilar, S., Pessina, P., Serrano, A.L., Muñoz-Cánoves (2011). Aberrant repair and fibrosis development in skeletal muscle. Muscle Nerve. 1: 21. 5) Vandesompele, J., De Preter, K., Pattyn, F., Poppe, B., Van Roy, N., De Paepe, A., et al. (2002). Accurate normalization of real-time quantitative RT-PCR data by geometric averaging  of multiple internal control genes. Genome Biol. 3, RESEARCH0034.    Take home message  Proliferation of Duchenne muscular dystrophy fibroblasts is reduced by methyl-prednisolone through the modulation of NFAT5 localization in the cell (by trapping in the cytoplasm). Affiliations:In IF: NFAT5 ( conclusion Introduction  Duchenne muscular dystrophy (DMD) is characterized by chronic inflammation and impaired muscle regeneration (Abdel-Salam et al., 2009). The major debilitating factor in DMD is the formation of fibrotic scar tissue (Banker and Engel, 2004). This scar tissue is an excess in extracellular matrix formation (ECM), especially collagen. Two major components induce fibrosis: chronic inflammation and chronic proliferation of fibroblast-like cells (Mann et al., 2001).   The major cell type involved in fibrosis is the fibroblast, producer of collagen. Fibroblasts are sensitive to several cytokines. CD4+ Th1 cells and CD8+ T cells produce IFN-γ, that exerts a pro-fibrotic role by mediating increased TNF-α production in macrophages. Amongst other cytokines, fibroblasts react to IL-1β and produce it in turn (autocrine function), inducing proliferation and ECM production (Kendall and Feghali-Bostwick, 2014).     Material and methods  DMD fibroblasts were cultured in vivo        Immunofluorescence microscopy (IF): Addition of cytokines for 24 h or 15 d • NFAT5 goat polyclonal (1/100, Santa Cruz, USA).  Western blotting (WB):  • NFAT5 mouse (1/100) (Santa Cruz Biotechnology, Texas, USA). • Tubuline  (1/1000)  (Sigma Aldrich, USA). RT-qPCR:  following the protocol of Vandesompele  et al., 2002) IncuCyte ZOOM: (Essen Bioscience, Hertfordshire,UK). The IncuCyte Software acquired and analyzed data at fixed points in each well. (4 images /well every 2 h for 15 d). Results           Immunofluorescence microscopy (IF):   DMD after 24 h   DMD after 15 d  50 µm   control   cytokines  cytokines    + methyl-    predniso-    lone IF: NFAT5 (red) + DAPI in nuclei (blue) merged  reveal NFAT5 being localized in the peri-nuclear  area after 24 h (white arrows) and shows decreased  NFAT5 staining  per cell after 15 d in DMD  fibroblasts exposed to cytokines + methyl- prednisolone (n=3 passages) (p<0.003). A part of the cultures received a clinical dose of methylprednisolone every other 3 days during 15 d:  0,75 mg/kg Western blotting:             1              0.29               0.70       1                   1.33                 0.87              DMD after 24 h    DMD after 15 d Densitometric  Analysis NFAT5/tubuline NFAT5     Tubuline        Tubuline NFAT5 WB:Total NFAT5 protein amounts decreased after 15 d exposure to  cytokines + methylprednisolone  (MP) (n=3 passages).   RT-qPCR: Relative NFAT5 mRNA expression  after  15 days treatment Relative NFAT5 mRNA expression  after 24 h treatment  * (1)              6   4   2 Relative NFAT5  mRNA expression + SD   1   =  control             =  cytokines          =  cytokines +                       methylprednisolone       RT-qPCR: Relative mRNA NFAT5  expression is not significantly  decreased after 15 d exposure to  cytokines + methylprednisolone  (n=1 passage).                Results and conclusion           Incucyte: Statistics:  SPSS 25.0 (IBM, Armonk, New York, USA).     100      80      60      40       20     0                  24 h                                   48 h                                         72 h                                       96 h                                      120 h                           144 h                                   168 h                                             192 h                                              360 h                    Cell confluence  (%) * *=   p<0.05     100       80       60       40       20         0 * Incucyte:  After 15 d, DMD fibroblasts exposed to cytokines + methyl- prednisolone (MP) show less confluence  (p <0.05) (n=3 passages).   Cell confluence  (%) DMD fibroblasts show reduced NFAT5 staining, decreased NFAT5 protein expression  and reduced proliferation after 15 d exposure to cytokines + methylprednisolone  (n=  3 passages).           =control   =cytokines   =cytokines              + MP            

The proliferation of Duchenne muscular dystrophy fibroblasts cultured under inflammatory conditions is reduced by methylprednisolone through modulation of NFAT5 localization in the cell

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The proliferation of Duchenne muscular dystrophy fibroblasts cultured under inflammatory conditions is reduced by methylprednisolone through modulation of NFAT5 localization in the cell

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