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SAT0066 NF-κB signaling in rheumatoid arthritis drives inflammation and joint destruction in part through inactivation of forkhead box class O transcription factors
  1. L.M. Hartkamp1,2,
  2. A.M. Grabiec1,2,
  3. J. Ludikhuize1,
  4. I.E. van Es1,2,
  5. P.P. Tak1,
  6. K.A. Reedquist1,2
  1. 1Clinical Immunology and Rheumatology
  2. 2Experimental Immunology, AMC, Amsterdam, Netherlands


Background Rheumatoid arthritis (RA) stromal fibroblast-like synoviocytes (FLS) are key effector cells in the pathology of RA, and share many phenotypic characteristics with transformed cancer cells. In many cancer models, inflammatory NF-κB signaling promotes transformation via inactivation of forkhead box class O (FoxO) transcription factors, classically regulated by phosphatidylinositol 3-kinase (PI3-K).

Objectives As both NF-κB and PI3-K pathways are activated in RA synovial tissue, we examined potential functional interactions between these pathways.

Methods Expression and/or phosphorylation of IKKβ, IκBα, PTEN, a negative regulator of PI3-K, protein kinase B (PKB) and FoxO1, downstream targets of PI3-K, was detected by immunohistochemistry combined with digital image analysis in synovial tissue from 15 disease-modifying antirheumatic drug (DMARD)-naive RA patients. Biopsies were obtained at baseline, and x-rays made of hands and feet at baseline and at 2 years follow-up. At 2 years, patients were classified as having non-erosive or erosive disease (Sharp van der Heijde score >2). Effects of PI3-K isoform specific inhibitors on RA FLS IL-1β- induced IκBα phosphorylation and NF-κB p65 activation were assessed by immunoblotting and ELISA, respectively. Furthermore, effects of NF-κB inhibition on LPS induced FoxO nuclear export was assessed by immunoblotting. RA FLS were transduced with adenovirus encoding control GFP or constitutively active FoxO1ADA to examine the effects on RA FLS NF-κB dependent gene sets using low density qPCR arrays.

Results In vivo we observed a strong correlation between expression of IKKβ and phosphorylation of its target IκBα (R=0.70, p<0.01). Similarly, activation of PKB correlated with phosphorylation of FoxO1 (R=0.66, p<0.01). Reciprocal positive correlations were also observed between activation of NF-κB and PI3-K pathways. In vitro, pan-PI3-K inhibitor LY294002 and inhibitors specific for α/β, γ and δ isoforms of PI3-K failed to influence IL-1β and LPS-induced activation of NF-κB transcriptional activity and cytokine production. However, inhibitors of IKKβ significantly reduced nuclear exclusion of FoxO proteins in FLS and macrophages, and increased FoxO DNA-binding activity. qPCR array analysis of NF-κB-dependent gene sets induced by IL-1β in RA FLS identified several genes, including CCL2, ICAM1 and BCL2A1, which were suppressed by introduction of constitutively active FoxO1.

Conclusions Our data suggest that NF-κB signaling drives inflammation and joint destruction in RA at least in part via inactivation of FoxO transcription factors.

Disclosure of Interest None Declared

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