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

Abstract

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-γ, 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-γ, 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.

Store-Dependent Ca2+ Entry in Endothelial Progenitor Cells As a Perspective Tool to Enhance Cell-Based Therapy and Adverse Tumour Vascularization

“…no. WO2005/009954 and US2004/ 02379; Synta Pharmaceuticals) [162,196]. Synta 66 was found to inhibit SOCE with an IC 50 value of 3 μM in gut T cells and was negatively probed against a large panel of additional ion channels, including voltage-dependent Na + channels, VGCCs, and inward rectifier K + channels, but not TRP conductances [196].…”

“…WO2005/009954 and US2004/ 02379; Synta Pharmaceuticals) [162,196]. Synta 66 was found to inhibit SOCE with an IC 50 value of 3 μM in gut T cells and was negatively probed against a large panel of additional ion channels, including voltage-dependent Na + channels, VGCCs, and inward rectifier K + channels, but not TRP conductances [196]. A recent investigation demonstrated that Synta 66 inhibited Orai1-gated Ca 2+ influx with a significantly higher potency (IC 50 of approximately 25 nM) in both human umbilical vein ECs (HUVECs) and in circulating ECFCs [24].…”

Molecular pharmacology of store-operated CRAC channels

“…138 In another study, anti-CD3/CD28 stimulated mononuclear cells isolated from the lamina propria of patients with Inflammatory bowel disease showed reduced cytokine production and T-bet transcription factor activation when pre-treated with Synta66, suggesting a potential role for CRAC channels in this chronic inflammatory state. 140 An increasing number of studies are employing Synta66 for investigating the physiological role of CRAC channels in vitro and animal models; however, its mechanism of action and specificity for the different Orai isoforms remain poorly understood.…”

TCAIM Decreases T Cell Priming Capacity of Dendritic Cells by Inhibiting TLR-Induced Ca2+ Influx and IL-2 Production

“…Ca 2+ influx is induced by certain but not all microbial products, such as LPS, CpG, or peptidoglycan, resulting in a calcineurin-dependent NFAT activation, subsequent nuclear translocation, and NF-kB activation by degradation of the NF-kB inhibitor IkB (31). Applying CRAC channel inhibitors results in a less mature phenotype (32) and a reduced IL-2 secretion (33).…”