TNFα blockade inhibits T cell cycling by NOTCH-1 signaling

Introduction: Therapeutic TNFα blockade, such as infliximab (IFX) or adalimumab (ADA) is gaining acceptance in inflammatory diseases, such as Crohn's disease (CD) and ulcerative colitis (UC). However, induction of apoptosis is not an obligation for TNFα inhibitors to act. Thus, further mechanisms of action for TNFα inhibitors must be uncovered.

Methods: Mononuclear cells (PBMC) from control, CD, and UC patients were activated by anti-CD3 antibodies and cultured with IFX or ADA. To examine signaling pathways, cells were cultured in the presence of TACE inhibitors (TAPI), TNFα receptor antibodies, and Notch ligand (Jagged). Furthermore, small interfering (si) RNA for TNFα and Notch-1 were also employed to block protein synthesis. After 48 hours, cells were examined using flow cytometry for activation (CD25), cell cycling (cyclin B1), apoptosis (Annexin-V/PI), Notch-1 and membrane (tm) TNFα expression. Immunoblotting (IB) was performed to determine cell cycle regulators and Notch-1 expression.

Results: Both IFX and ADA significantly inhibited activation, cell cycling and expansion of stimulated PBMC. Moreover, in all populations investigated, ADA and IFX inhibited protein expression of tmTNFα, Retinoblastoma phosphorylation, and up-regulated cell cycle inhibitor p21 expression; confirming the central role of TNFα in T cell cycling. Also inhibition of TNFα synthesis by siRNA prevented anti-TNFα induced restriction of T cell activation. As Notch links cell cycling and apoptosis, we tested weather Notch might be the link by which TNFα inhibitors regulate T cell cycling. Indeed, both ADA and IFX notably induced Notch-1 expression. Also, vice versa, silencing Notch-1 averted the anti TNFα-induced T cell cycle arrest. FACS staining confirmed that IFX and ADA induced T cell apoptosis. Interestingly, inhibition of TACE, completely abolished anti-TNFα-induced apoptosis.

Conclusion: Our study uncovered that blockade of TNFα signaling by silencing or TNFα blockers potently inhibits activation and cell cycling of normal and IBD T cells. By uncovering that NOTCH-1 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 signaling pathway by which biologicals act in IBD.