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
Authors: K. Umekita, S. Miyauchi, M. Matsuda, K. Kubo, M. Komura, H. Nomura, A. Kawano, K. Umeki, I. Takajo, Y. Nagatomo, M. Frank-Bertoncelj, R.E. Gay, S. Gay, A. Okayama
Journal: Annals of the Rheumatic Diseases (2016)

<h3>Background</h3> 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. <h3>Objectives</h3> To clarify the molecular mechanisms of TLR-4 signaling pathways in activated RASF. <h3>Methods</h3> 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. <h3>Results</h3> 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. <h3>Conclusions</h3> 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. <h3>References</h3> 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) <h3>Acknowledgement</h3> We thank Ms. Yuki Kaseda, Ms. Ayaka Miyamoto and Dr. Yatsuki Aratake for their excellent technical assistance. <h3>Disclosure of Interest</h3> None declared