IL-6 and TNF are Potential Inflammatory Biomarkers in Facioscapulohumeral Muscular Dystrophy

Figures

• 

  Fig. 1. Study Workflow. (1) As a first step a selected group of markers (IL-6, TNF, IL-1α, IL-1β, MCP-1, and VEGF-A) were measured in serum samples of FSHD patients and sex- and age matched healthy control using the highly sensitive multi-analyte Ella Simple Plex Cartridge Kit. (2) Next, whole blood immunophenotyping and trained immunity of monocytes was studied in FSHD patients and matched healthy controls. (3) Third, trained immunity and ex vivo stimulation with TLRs ligands was studied in NK cells of FSHD patients and matched healthy controls. (4) Lastly, we characterized inflammation not only at a systemic but also at a local level using the highly targeted MRI-guided muscle biopsies. 21 pair of FHSD MRI guided muscle biopsies (TIRM+ and TIRM–) and 8 healthy control muscle biopsies were ex vivo stimulated for 24 hours.

• 

  Fig. 2. Serum circulating inflammatory markers. The concentrations (pg/ml) of a selected group of circulating inflammatory markers were measured in sera samples of patients with FSHD and sex and age matched healthy controls. (A) Mean±SD of IL-6 measured in serum samples of 150 patients with FSHD and 98 sex and age matched healthy controls. (B) Mean±SD of TNF measured in serum samples of 150 patients with FSHD and 59 sex and age matched healthy controls. (C) Mean±SD of IL-1α measured in serum samples of 150 patients with FSHD and 66 sex and age matched healthy controls. (D) Mean±SD of IL-1β measured in serum samples of 150 patients with FSHD and 98 sex and age matched healthy controls. (E) Mean±SD of MCP-1 measured in serum samples of 14 patients with FSHD and 14 sex and age matched healthy controls. (F) Mean±SD of VEGF-A measured in serum samples of 14 patients with FSHD and 14 sex and age matched healthy controls. Groups were compared with the non-parametric Mann Whitney test. The obtained raw p-values were adjusted after multiple correction with Bonferroni test. *p < 0,05, **p < 0,005, ***p < 0,001.

• 

  Fig. 3. Whole blood immunophenotyping. EDTA blood samples of 15 patients with FSHD and 15 sex and age matched healthy controls were stained for 30 minutes in dark at 4°C with the following fluorophore-conjugated antibodies cocktail: CD45, CD14, CD16, CD3, CD56, CD19, CD4, CD8, CD25. Samples were analysed on a 10-color Navios flow cytometer (Beckman Coulter). (A) Mean±SD of percentages of total leukocytes (CD45+), total neutrophils, and total monocytes (CD14+). (B) Mean±SD of classical (CD14++CD16–), non-classical (CD14++CD16+), and intermediate monocytes (CD14+CD16+). (C) Mean±SD of T-Cells CD4+, T-Cells CD8+, and T-Cells CD25+ (T-Reg). (D) Mean±SD of NK CD56+CD16–, CD56++CD16–, and CD56+CD16+. Groups were compared with the non-parametric Mann Whitney test.

• 

  Fig. 4. Ex vivo β-glucan induction of trained immunity in monocytes and NK cells of patients with FSHD and sex and age matched healthy controls. (A) Monocytes and NK cells of patients with FSHD and healthy controls were trained with RPMI (–), taken as negative control, or β-glucan (+) for 24 hours. On day 6, monocytes differentiated into macrophages and NK cells were secondary stimulated with either RPMI or LPS for 24 hours. (B-C) Mean±SD of IL-6 and TNF concentration (pg/ml) measured in the supernatant of LPS restimulated monocytes derived macrophages of 15 patients with FSHD and 15 sex and age matched healthy controls. (D-E) Mean±SD of IL-6 and TNF concentration (pg/ml) measured in the supernatant of LPS restimulated NK cells of 11 patients with FSHD and 11 sex and age matched healthy controls. Groups were compared with the non-parametric Mann Whitney test. The obtained raw p-values were adjusted after multiple correction with Bonferroni test. *p < 0,05.

• 

  Fig. 5. NK cells 24 h ex vivo TLRs ligands stimulation and cytotoxicity activity. (A) Graphical representation of experimental set-up. 1) NK cells of 39 patients with FSHD and 22 sex and age matched healthy controls were ex vivo stimulated with RPMI (cell medium), LPS (lipopolysaccharide), P3C (Pam3Cys), and Candida (C. albicans Heat Killed) for 24 hours. 2) As a second step, NK cells cytotoxicity assay was performed in a subset of 7 patients with FSHD and 7 sex and age matched healthy controls. NK/K562 target ratio 1:1 and NK/K562 target ratio 2:1 was stained with Ann V, PI, CD107a, and Granzyme B after 4 hours of co-incubation (B) Mean±SD of IL-6 and TNF concentration (pg/ml) measured in the supernatant of TLRs stimulated NK cells. (C) Percentage of target cells showing uptake of Ann V only, PI only, and Ann/PI respectively. (D) Percentage of NK cells showing expression of CD107a and Granzyme B following stimulation with K562 cell at 1:1 ratio or stimulation with K562 cells at a 2:1 ratio Groups were compared with the non-parametric Mann Whitney test. The obtained raw p-values were adjusted after multiple correction with Bonferroni test. *p < 0,05; **p < 0,005.

• 

  Fig. 6. Whole muscle ex vivo stimulation. (A) Representative TIRM and DIXON MRI of a 62-year-old patient with FSHD1. (A1) MRI cross section of the right upper leg showing TIRM hyperintensity of gastrocnemius medialis. (A2) Corresponding DIXON-MRI cross section showing degree of muscle fatty infiltration. (A3) MRI cross section of the right upper leg showing TIRM normointensity of vastus lateralis. (A4) Corresponding DIXON-MRI cross section showing normal appearing muscle. The black arrow indicates the site of the TIRM+ biopsy. The white arrow indicates the site of the TIRM- biopsy. (B) Mean±SD of IL-6 and TNF concentration (pg/ml) from supernatant of 21 stimulated matched pairs of TIRM+ and TIRM–whole muscle specimens. (C) Mean±SD of IL-6 and TNF concentration (pg/ml) from supernatant of 21 ex vivo stimulated TIRM+ whole muscle specimens and 8 healthy control whole muscle specimens. (D) Mean±SD of IL-6 and TNF concentration (pg/ml) from supernatant of 21 ex vivo stimulated TIRM–whole muscle specimens and 8 HC whole muscle specimens. Groups were compared with either the non-parametric Wilcoxon matched pairs test (B) or the non-parametric Mann Whitney test (C-D). The obtained raw p-values…

• 

  Fig. 7. Representative immuno-histological analysis of muscle biopsies. (A) HPhlox staining and immunostaining (CD3, CD4, CD8, CD56, CD68, and HLA-ABC respectively) of serial muscle cryo-sections from a TIRM+ MRI guided muscle biopsy of a 62-year-old patient with FSHD1. The HPhlox staining shows the variability of the fiber size along the section, increased central internal nuclei, the presence of few regenerating fibers, mild local fibrosis and perivascular inflammatory infiltrates. Immunostaining shows that inflammatory infiltrates comprised predominantly CD3, CD8, and CD68 positive cells. CD56 positive cells can be also appreciated at the same inflammatory sites. The staining for HLA-ABC shows evident upregulation. (B) Representative HPhlox and immunostaining (CD3, CD4, CD8, CD56, CD68, and HLA-ABC respectively) of serial muscle cryo-sections from the corresponding TIRM–MRI guided muscle biopsy of the same patient. The HPhlox staining shows normal findings and no muscular dystrophic changes. Along with a normal muscle histology, no inflammatory infiltrates were detected on additional immunostaining and no HLA-ABC upregulation was appreciated. (C) Representative HPhlox and…