Fig. 5: TNF inhibits St6gal1 expression to down-regulate α2-6-sialylation in synovial fibroblasts.

a Illustration shows pathways involved in sialic acid and sialylated glycans biosynthesis. Relative expression for each gene was extracted from bulk RNA-Seq data comparing naive and arthritic SFs directly isolated from mouse synovium. b Synovial fibroblasts expanded from healthy or arthritic mouse joints were stimulated in vitro for 6 h with recombinant IL-1β, TNF, or IL-17A [10 ng/ml]. mRNA expression for genes involved in sialylation [as shown in a] was evaluated by RT-qPCR. Il6 was included as a positive control to confirm cell activation. Volcano plots show log2 fold difference in stimulated cells (x axis) and p-value (y axis). Each dot represents the mean of three independent experiments analyzed in triplicate, with naive cells in blue and arthritic cells in red. The pink line indicates a twofold-change in gene expression threshold and blue lines indicate p = 0.05 threshold in the t-test to evaluate statistical significance. c TNF, but not IL-1β or IL-17A reduces α2-6-sialylation of synovial fibroblasts both at RNA (St6gal1 expression) and protein (SNA binding) levels. Expression of St6gal1 (left panel) was evaluated by RT-qPCR. Results show the mean of three independent experiments analyzed in triplicate, error bars represent SEM. Statistical significance was determined using two-tail unpaired t-tests where *p < 0.05 and **p < 0.01. Actual p-values: a 0.0069, b 0.0277. Expression of α2-6 sialylated glycoconjugates was determined by SNA binding (right panel) as in Fig. 4a. Results are merged from 3 (naive) and 2 (CIA) independent experiments, n = 10 (DMEM naive), 16 (IL-1 naive and IL-17 naive), 13 (TNF naive), 10 (DMEM CIA), 12 (IL-1 naive and IL-17 naive), and 9 (TNF CIA). Statistical significance was determined using two-tail unpaired t-tests, where **p < 0.01 and ***p < 0.001. Actual p-values: c 0.0124, d 0.0091. d SFs were expanded from OA human synovium. Cells were stimulated with recombinant human TNF (10 ng/ml) for 6 h. IL6, ST6GAL1, and ST6GAL2 expression were determined by qPCR. Results show relative expression to HPRT, showing mean ± SEM. Statistics: one-tail unpaired t-test, n = 3 biological replicates from cells pooled from three donors, ***p < 0.001, *p < 0.05. Actual p-values: e <0.0001, f 0.0170, g 0.0321. e Relative expression of individual sialylated glycan structures was evaluated by MALDI-TOF MS analysis as in Fig. 3. Ratios of sialylated vs non-sialylated twin structures were evaluated for non-stimulated cells (blue) and TNF stimulated (red) cells (48 h, 10 ng/ml). Results are from one single experiment using pooled cells from three animals.