Fig. 9: Synovial fibroblasts desialylation triggers inflammatory cytokine production.

a Murine SFs isolated from healthy mice were treated with either siRNA for St6gal1 or Allstars RNA control for 3 days. The reduction of α2-6-sialic acid was evaluated by SNA binding by immunofluorescence and flow cytometry. Expression of St6gal1 mRNA and Il6 mRNA was determined by qPCR. b SFs were treated with St6gal1 siRNA or Allstars RNA for 3 days. Cell culture medium was then replaced and cells were incubated for 12 h at 37 °C. Cytokine concentration was determined by ELISA and data were normalized with the number of cells in each well, calculated by crystal violet staining. Each dot represents one experiment analyzed in technical triplicates. Statistical significance was evaluated by ordinary one-way ANOVA and Tukey’s test for multiple comparations, *p < 0.05, **p < 0.01. Actual p-values: a 0.0474, b 0.025, c 0.100. c murine SFs were incubated with 100 mU/ml of recombinant Clostridium perfringens (CP) sialidase for 30 min at 37 °C. Cells were then washed and fixed. Relative lectin binding (SNA, MAA, and AAL) was then calculated by ELLA assay. Data are from one representative experiment performed in five biological replicates. The experiment was repeated four times with similar results. ***p < 0.001 analyzed by one-tail t-test. Actual p-values: d 0.0003, e <0.0001. d Synovial fibroblasts were treated with 100 mU/ml of recombinant Clostridium perfringens (CP) sialidase as before and incubated for 3 h at 37 °C. RNA was then isolated to quantify relative expression levels of Il6, Ccl2, and Mmp3 by qPCR. Data represent fold change expression compared to non-treated controls. Each dot represents one independent experiment analyzed in triplicate, data are presented as mean ± SEM for n = 5 experiments for Il6 and Ccl2 and n = 4 experiments for Mmp3. A two-tail paired t-test using relative expression was used to calculate statistical significance. *p < 0.05, **p < 0.01. Actual p-values: f 0.0034, g 0.0265.