Background/Purpose: A comprehensive epigenomic characterization of rheumatoid arthritis (RA) fibroblast-like synoviocyte (FLS) has recently been described and can help to identify new critical pathways for RA pathogenesis. Two genes involved in sialylation, β-galactoside α2,3-sialyltransferase I (ST3Gal1) and β-galactoside α2,6-sialyltransferase I (ST6Gal1), were found to be significantly associated with changes in epigenetic marks between RA and osteoarthritis (OA) FLS, suggesting a role of this biological process in the aggressive phenotype of RA FLS. Here, we determined whether the amount of sialic acid (Sia) on FLS regulate its phenotype.

Methods: Methods: Sia expression in OA and RA synovial tissue was determined by using lectin immunofluorescence (IF).  Sialyltransferases (ST) gene expression and Sia expression after TNF or PDGF stimulation in RA FLS were analyzed by qPCR and by using lectin western blotting (WB),respectively.  Migrative (scratch assay) and invasive (matrigel assay) phenotype after PDGF stimulation were evaluated after treatment with neuraminidase (Neu) from Clostridium perfringens (25mU/ml for 60 minutes), which cleaves Sia from glycoconjugates, and with an endogenous neuraminidase inhibitor, N-Acetyl-2,3-dehydro-2-deoxyneuraminic acid (NADNA, 100uM). Metalloproteinases (MMP) and IL6 gene expression after TNF and PDGF stimulation was also analyzed after Neu treatment. For arthritis experiments, mice were injected with K/BxN sera on day 0. Lith-O-Asp (a sialyltransferase inhibitor, 3mg/kg) was injected every other day i.p. beginning on day 0 after serum administrationor starting at the peak of arthritis (from day 4). Clinical arthritis scores were serially assessed.

Results: Results:Maackia amurensis lectin I and II IF staining was higher in RA synovial lining.PDGF and TNF stimulation increased the expression of ST3Gal1 and ST6Gal1 and total amount of Sia in FLS. Neu treatment decreased the amount of Sia in FLS determined by WB and inhibited not only migration (Neu:26.94±5.4vs. vehicle:22.5±3.4; p< 0.001) and invasion (Neu:12.7±1.6 vs. vehicle:24.0±2.5; p< 0.001), but also the expression of MMP and IL-6 after TNF and PDGF stimulation.  Conversely, NADNA treatment increased the amount of Sia in RA FLS and enhanced both migration (vehicle:32.0±3.1vs. NADNA:16.5±5.0; p< 0.001)and invasion (vehicle:27.0±9.3vs. NADNA:39.5±13.6; p< 0.001). Finally, Lith-O-Asptreatment significantly decreased arthritis severity. Day 10 scores were 15.16±1.17 and 12.3±1.4(p=0.003) for vehicle and Lith-O-Asp-treated mice respectively, when mice were treated from day 0, and 15.2±1.2 and 11.3±2.2(p< 0.001) for vehicle and Lith-O-Asp -treated mice when mice were treated from day 4.

Conclusion: Conclusion: Sia are abundant on vertebrate glycoproteins and mediate a variety of biological phenomena, including migration and invasion. Our results suggest that in FLS, changes in the amounts of Sia likely mediated by changes in expression of ST3Gal1 and/or ST6Gal1, might be key regulator of FLS phenotype and contribute to joint destruction in RA. Modulation of Sia expression could act as disease modifying factor by directly modulating synoviocyte-mediated cartilage destruction.

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ACR Meeting Abstracts - https://acrabstracts.org/abstract/role-of-sialic-acid-in-the-aggressive-phenotype-of-ra-fls/