N.K.VERMA1, M.H.U.T. FAZIL1, C.S. CHIRUMAMILLA2, C. PEREZ-NOVO2, B.H.S. WONG1, S. KUMAR3, S.K. SZE1, W.V. BERGHE2
Nanyang Technological University1, University of Antwerp2, Indian Agricultural Statistics Research Institute3
Withaferin A (WFA) is a dietary phytochemical derived from Withania somnifera and exhibits a wide array of biological properties. Here we investigated the effect of WFA on T-cell migration and cytokine production, which are crucial for adaptive immune responses as well as skin inflammatory reactions.
Human primary and cultured T-cells were stimulated to migrate via the engagement of the integrin LFA-1 receptor to its ligand. Standard molecular and imaging assays, including proteomics, kinomics, Western immunoblotting, and confocal microscopy were performed.
WFA dose-dependently (within the concentration range of 0.3 to 1.25 μM) inhibited the ability of T-cells to migrate and produce inflammatory cytokines IL-2 and IFN-γ (>80% inhibition). Co-immunoprecipitation with subsequent tandem mass-spectrometry detected 273 unique proteins as WFA intracellular targets. Further protein network analysis identified the ZAP70 kinase as a key WFA-binding protein. Phospho-peptide mapping and kinome analysis substantiated kinase signaling downstream of ZAP70 as WFA targets, which was validated by in silico docking of the ZAP70 protein and the use of a WFA analogue withanone. WFA impaired LFA-1-induced phosphorylation of ZAP70 at the tyrosine 319 residue (>50% inhibition). The WFA-ZAP70 interaction was disrupted by a redox agent dithiothreitol, suggesting a covalent binding interface. Moreover, WFA ablated the serine 19 phosphorylation of the cytoskeletal myosin light chain protein, further constraining T-cell motility.
We present a novel mechanism whereby WFA inhibits T-cell motility and cytokine production. WFA can therefore be exploited to pharmacologically redirect cytokine imbalance and control T-cell trafficking in chronic inflammatory conditions.