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AB0027 A Novel Transcription Factor NFAT5 Plays An Important Role as Critical Regulator in The Inflammatory Response of Rheumatoid Arthritis Fibroblasts Mediated via Toll-Like Receptor 4 Signaling Pathways
  1. K. Umekita1,
  2. S. Miyauchi1,
  3. M. Matsuda1,
  4. K. Kubo1,
  5. M. Komura1,
  6. H. Nomura1,
  7. A. Kawano1,
  8. K. Umeki1,
  9. I. Takajo1,
  10. Y. Nagatomo1,
  11. M. Frank-Bertoncelj2,
  12. R.E. Gay2,
  13. S. Gay2,
  14. A. Okayama1
  1. 1Division of Rheumatology, Infectious Diseases and Laboratory Medicine, Faculty of Medicine, University of Miyazaki, Miyazaki, Japan
  2. 2Center of Experimental Rheumatology, University Hospital Zurich, Zurich, Switzerland

Abstract

Background Damage-associated molecular patterns (DAMPs) are proposed to drive aberrant stimulation of Toll-like receptors (TLRs) in the rheumatoid arthritis (RA) joints resulting in increased expression of proinflammatory cytokines and chemokines. In the recent study we demonstrated that the neutrophil-derived lactoferrin (LTF) induces inflammatory response in RA synovial fibroblasts (RASF) via TLR-4 (Ref.). However, the molecular mechanisms of TLR-4 signaling in activated RASF are still unclear.

Objectives To clarify the molecular mechanisms of TLR-4 signaling pathways in activated RASF.

Methods Recombinant human neutrophil-derived LTF was used as one of the TLR-4 ligands. RASF were treated with LTF and/or TNF-α, and the expression of proinflammatory cytokines and chemokines, such as IL-6, IL-8 and CCL20 in RASF was measured by RT-qPCR and ELISA. To repress the TLR-4 signaling pathways, a small molecular inhibitor of TLR-4 (TAK-242), TAK1 inhibitor (5Z-7-Oxozeaenol), nuclear factor kappa B (NF-kB) inhibitor (BMS345541), and p38 mitogen activated protein kinase (MAPK) inhibitor (SB202190) were used. The role of nuclear factor of activated T cells 5 (NFAT5) in the TLR-4 signaling in RASF was investigated using a small interfering RNA targeting NFAT5.

Results Stimulation of RASF with LTF significantly increased the expression of IL-6, IL-8 and CCL20 mRNA and proteins (p=0.01). LTF enhanced the expression of these cytokine and chemokine mRNA in RASF stimulated by TNFα. TAK-242 completely repressed the expression of these cytokines and chemokines in RASF stimulated by LTF, while the TAK-1 inhibitor did not suppress the expression of these cytokines and chemokines in RASF. The NF-kB inhibitor, but not the p38MAPK inhibitor, partially repressed the expression of IL-6 and IL-8 mRNA induced by LTF. However, neither the NF-kB inhibitor nor p38MAPK inhibitor repressed the expression of CCL20 mRNA. Interestingly, silencing of NFAT5 significantly decreased the basal expression of IL-6, IL-8 and CCL20 mRNA in RASF. Additionally, silencing of NFAT5 significantly repressed the expression of not only IL-6, IL-8, but also of CCL20 mRNA in RASF treated by LTF.

Conclusions These findings suggest that NFAT5 plays an important role as a critical regulator in the proinflammatory response of RASF mediated by the TLR-4 signaling pathway.

  1. Umekita K, et al. Neutrophil-Derived Lactoferrin Regulates the Activity of NFAT5 in Rheumatoid Arthritis Synovial Fibroblasts Via Toll-like Receptor 4. 2015 ACR/ARHP Annual Meeting (San Francisco)

Acknowledgement We thank Ms. Yuki Kaseda, Ms. Ayaka Miyamoto and Dr. Yatsuki Aratake for their excellent technical assistance.

Disclosure of Interest None declared

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