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This Article Full Text Full Text (PDF) Data Supplement All Versions of this Article: jimmunol.0802887v1 183/5/3454    most recent Alert me when this article is cited Alert me if a correction is posted Citation Map Services Similar articles in this journal Similar articles in PubMed Alert me to new issues of the journal Download to citation manager Request Permissions Google Scholar Articles by Di Sabatino, A. Articles by Kruidenier, L. PubMed PubMed Citation Articles by Di Sabatino, A. Articles by Kruidenier, L.

Targeting Gut T Cell Ca²⁺ Release-Activated Ca²⁺ Channels Inhibits T Cell Cytokine Production and T-Box Transcription Factor T-Bet in Inflammatory Bowel Disease

Antonio Di Sabatino^*,||, Laura Rovedatti^*,||, Rejbinder Kaur, Jonathan P. Spencer, Jon T. Brown, Valerie D. Morisset, Paolo Biancheri^*,||, Nicholas A. B. Leakey^*, Jonathan I. Wilde^¶, Laurie Scott^¶, Gino R. Corazza^||, Kevin Lee, Neel Sengupta, Charles H. Knowles, Martin J. Gunthorpe, Peter G. McLean, Thomas T. MacDonald^1,2,* and Laurens Kruidenier^1,

^* Centre for Infectious Disease, Centre for Academic Surgery, Institute of Cell and Molecular Science, Barts and the London School of Medicine and Dentistry, London, Immuno-Inflammation Centre of Excellence for Drug Discovery, GlaxoSmithKline, Stevenage, Neurology Centre of Excellence for Drug Discovery, GlaxoSmithKline, Harlow, ^¶ Discovery Technology Group, Molecular Discovery Research, GlaxoSmithKline, Harlow, United Kingdom; and ^|| First Department of Medicine, Fondazione Istituto Di Ricovero e Cura a Carattere Scientifico Policlinico S. Matteo, University of Pavia, Pavia, Italy

Prolonged Ca²⁺ entry through Ca²⁺ release-activated Ca²⁺ (CRAC) channels is crucial in activating the Ca²⁺-sensitive transcription factor NFAT, which is responsible for directing T cell proliferation and cytokine gene expression. To establish whether targeting CRAC might counteract intestinal inflammation, we evaluated the in vitro effect of a selective CRAC inhibitor on T cell cytokine production and T-bet expression by lamina propria mononuclear cells (LPMC) and biopsy specimens from inflammatory bowel disease (IBD) patients. The inhibitory activity of the CRAC blocker was investigated through patch-clamp experiments on rat basophilic leukemia cells and fluorometric imaging plate reader intracellular Ca²⁺ assays using thapsigargin-stimulated Jurkat T cells and its detailed selectivity profile defined using a range of in vitro radioligand binding and functional assays. Anti-CD3/CD28-stimulated LPMC and biopsy specimens from 51 patients with IBD were cultured with a range of CRAC inhibitor concentrations (0.01–10 µM). IFN-, IL-2, IL-8, and IL-17 were analyzed by ELISA. T-bet was determined by immunoblotting. We found that the CRAC blocker concentration-dependently inhibited CRAC current in rat basophilic leukemia cells and thapsigargin-induced Ca²⁺ influx in Jurkat T cells. A concentration-dependent reduction in T-bet expression and production of IFN-, IL-2, IL-17, but not IL-8, was observed in IBD LPMC and biopsy specimens treated with the CRAC inhibitor. In conclusion, we provide evidence that the suppression of CRAC channel function may dampen the increased T cell response in the inflamed gut, thus suggesting a promising role for CRAC inhibitor drugs in the therapeutic management of patients with IBD.

The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked advertisement in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

¹ T.T.M. and L.K. contributed equally to this work.

² Address correspondence and reprint requests to Prof. Thomas T. MacDonald, Institute of Cell and Molecular Science, Barts and the London School of Medicine and Dentistry, Queen Mary University of London, Whitechapel, London E1 2AT, U.K. E-mail address: t.t.macdonald{at}qmul.ac.uk

³ Abbreviations used in this paper: IBD, inflammatory bowel disease; CD, Crohn’s disease; UC, ulcerative colitis; CRAC, Ca²⁺ release-activated Ca²⁺; I_CRAC, CRAC current; FLIPR, fluorometric imaging plate reader; LPMC, lamina propria mononuclear cell; RBL, rat basophilic leukemia; SOC, store-operated calcium.

⁴ The online version of this article contains supplemental material.