989 - The Pattern-Recognition Receptor NOD1 Promotes Production of Inflammatory Mediators in Different Cell Types of the Synovium in Rheumatoid Arthritis

Monday, November 7, 2011: 9:00 AM-6:00 PM
Hall F2 - Poster Hall (McCormick Place West)
Kazuhiro Yokota1, Toshihide Mimura2, Renate E. Gay1, Steffen Gay1, Christoph Kolling3 and Caroline Ospelt1, 1Center of Experimental Rheumatology, University Hospital Zurich, Zurich, Switzerland, 2Saitama Medical University, Saitama, Japan, 3Schultess Clinic, Zurich, Switzerland
Presentation Number: 989

Background/Purpose: We previously reported that the functional pattern-recognition receptor NOD1 is significantly increased in rheumatoid arthritis (RA) compared to osteoarthritis (OA) synovial tissues and that stimulation of RA synovial fibroblasts (SFs) with the NOD1 ligand induces expression of pro-inflammatory cytokines. Now, we looked at the expression and regulation of NOD1 in RASFs, OASFs, monocyte-derived macrophages (MDMs) and peripheral blood mononuclear cells (PBMCs) and analyzed its interaction with TLR pathways.

Method: Expression of NOD1 mRNA and protein in RASFs, OASFs, healthy control PBMCs and MDMs was analyzed by real-time PCR and flow cytometry. RASFs, MDMs and PBMCs were stimulated with TNF, IL-1Β, NOD1 ligand, TLR2 ligand, TLR3 ligand and TLR4 ligand. ELISA was used to quantitate protein levels of IL-6 and IL-1Β. Silencing of NOD1 was performed with transfection of RASFs siRNA targeting NOD1 mRNA by Amaxa Nucleofector Technology. Phosphorylation levels of interleukin-1 receptor-associated kinase 1 (IRAK1) were measured by western blotting.

Result: Basal expression levels of NOD1 mRNA (n=4-5) and protein (n=5-6) were not different in RASFs, OASFs, healthy control PBMCs and MDMs. Only stimulation with TLR3 induced the expression of NOD1 mRNA (2.67±0.29-fold, p<0.05) and protein (2.30±0.59-fold, p<0.05) in RASFs, but not in PBMCs or MDMs. Thus, NOD1 is constitutively similarly expressed among different cell types of the RA synovium, but is upregulated by TLR3 stimulation only in RASFs. Even though PBMCs (n=6) and MDMs (n=6) did not produce IL-6 or IL-1Β by NOD1 stimulation alone, there was a strong synergistic effect of NOD1 with TLR2 as well as TLR4 in the production of IL-1Β and IL-6 in these cells. Similar synergisms were found in the production of IL-6 by RASFs (n=6), showing that NOD1 and TLRs strongly synergize in the induction of pro-inflammatory effects in RASFs, MDMs and PBMCs. Most interestingly, IL-6 production induced by TLR2 ligand or IL-1Β stimulation was significantly decreased by silencing of NOD1 compared with that of controls in RASFs (n=6). Silencing of NOD1 significantly reduced the phosphorylation of IRAK1 induced by TLR2 stimulation (n=9).

Conclusion:In the current study we show a strong interaction of NOD1 with other pattern recognition receptors in different synovial cells. In addition to a synergistic response of the parallel activation of these receptors, our data indicate a role of NOD1 in the promotion of TLR2 and IL-1Β signaling pathways in RA.

Keywords: nod-like receptor (NLR), rheumatoid arthritis (RA) and synovial cells, synovial fluid

Disclosure: K. Yokota, None; T. Mimura, None; R. E. Gay, None; S. Gay, None; C. Kolling, None; C. Ospelt, None.