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P035. Linking TNFα inhibitors and Notch-1: Novel implications in inflammatory bowel diseases

L. Werner, D. Paclik, U. Berndt, A. Sturm

Charité Hospital, Berlin, Germany

Introduction and Aims: The anti-TNFα antibodies Infliximab (IFX) and Adalimumab (ADA) are highly effective in the treatment of Crohn's disease (CD) and ulcerative colitis (UC). As their mechanisms of action are beyond the induction of apoptosis, and not fully uncovered yet; our study aims to further investigate anti-TNFα's mode of action in the human immune system. Specifically, we hypothesized that Notch signaling mediates anti-TNFα action and thus aspired to identify Notch as a link by which anti-TNFα antibodies mediate their additional inhibitory functions in IBD.

Methods: PBMC from normal, CD and UC patients were isolated, activated by anti-CD3 Ab and cultured with or w/o IFX, ADA, TACE inhibitors (TAPI), TNFα receptor antibodies, Notch inhibitors and/or Notch ligands. Small interfering (si) RNA for TNFα and Notch1 were employed to block protein synthesis. After 48 h, FACS was performed to determine cell activation (CD25, CD80, CD86), trans-membrane (tm) Notch1, tmTNFα expression, cell cycling and apoptosis/necrosis (Annexin-V/PI). Immunoblotting was performed to determine Retinoblastoma (Rb), p21, p53, and Notch1 intracellular domain (NICD) expression. ELISA detected cytokine secretion.

Results: T cell stimulation induced pro-inflammatory cytokines secretion and tmTNFα expression. Both IFX and ADA prevented significantly this upregulation. Analyzing cell activation and cell cycling, ADA and IFX significantly inhibited CD25, CD80, and CD86 expression, Rb phosphorylation, and finally cell cycling and expansion. Regarding further cell cycle regulators; IFX and ADA up-regulated cell-cycle inhibitors p21 and p53 levels in stimulated normal, CD and UC PBMC. At the same time, IFX and ADA induced apoptosis of activated T cells in all groups investigated. Interestingly, inhibition of TNFα synthesis, TNFαR and TACE activity completely abolished anti-TNFα-induced apoptosis; but not their ability to decrease cell cycling. Linking Notch and TNFα, we showed that ADA and IFX abrogated activation-induced tmNotch1 with a concurrent increase of NICD, suggesting activation of Notch. Also, silencing of Notch1 almost completely averted the anti-TNFα-induced T cell cycle arrest.

Conclusion: IFX and ADA robustly inhibit T cell activation, cell cycling and expansion in normal and IBD T cells. By uncovering that Notch1 mediates the inhibitory effects of ADA and IFX on T cell cycling, we provide not only a new mode of action, but also an underlying signalling pathway by which biologicals act in IBD.