MECHANIMS INVOLVED IN THE INDUCTION OF IL-6 IN FIBROBLAST-LIKE SYNOVIOCYTES BY STIMULATION WITH SPONDYLOARTHRITIS SYNOVIAL FLUID

M.S. DI GENARO; E. CALLEGAR; R.BLAS; A. MUNARRIZ; R. PARDO-HIDALGO; H. TAMASHIRO

Buenos Aires

Congreso; Congreso Panamericano de Reumatologia; 2018

Sociedad Panamerica de Reumatología

Objectives: In previous studies we have reported that synovial fluid (SF) ofspondyloarthritis (SpA) patients induces interleukin (IL)-6 production in fibroblast-like synoviocytes (FLS), and this cytokine modulates metalloproteinase-9activity of these cells. However, the molecules of the SpA synovial microenvironmentthat trigger this pro-inflammatory circuit are unknown. The purposeof the present work was to examine the candidate endogenous molecules ofSpA SF, and the mechanisms involved in the induction of IL-6 in FLS.Methods: The SF of SpA patients were pooled (n=9). Human synovial fibroblastsof the SW982 cell line were stimulated with SpA SF pool at dilution1:10 in DMEM culture medium in absence or presence of anti-tumor necrosisfactor (TNF) (Infliximab, 200 g/ml), TNF inhibitor (Etanercept, 100 g/ml), anti-IL-17 (Sekukinumab, 100 g/ml), anti-IL-6 receptor (IL-6R) (Tocilizumab,200 g/ml), Toll-like receptor 4 (TLR4) activation inhibitor (Polymyxin B,50 g/ml), ERK, JNKor p38 mitogen-activated protein kinase (MAPK) inhibitor(10M), or Janus kinase (JAK) 1/3 inhibitor (Tofacitinib, 1000nM). SF treatedwith Hyaluronidase (50 g/ml) (H-SF) or Proteinase K (500 g/ml) (P-SF) was alsoused for stimulation of FLS. The IL-6 levels in the supernatants weremeasured bya commercial enzyme-linked immunosorbent assay (ELISA) kit. In addition, IL-6-inducter alarmins in SpA SF were identified by a Gel-free and Gel-LC botton-upmass spectrometry-based proteomics through the 2D-nano-liquid chromatographytandemmass spectrometry (2D-nano-LC-MS/MS) analysis. The data were analyzedby one-way analysis of variance (ANOVA) followed by Tukey multiplecomparison test. A p value ≤ 0.05 was considered statistically significant.Results: We found that anti-TNF, anti-IL-17 and anti-IL-6R significantly reducedIL-6 secretion by FLS (p