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.
Prolonged Ca(2+) entry through Ca(2+) release-activated Ca(2+) (CRAC) channels is crucial in activating the Ca(2+)-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 Ca(2+) 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 microM). IFN-gamma, 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 Ca(2+) influx in Jurkat T cells. A concentration-dependent reduction in T-bet expression and production of IFN-gamma, 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.
Di Sabatino
Antonio
A
Centre for Infectious Disease, Institute of Cell and Molecular Science, Barts and the London School of Medicine and Dentistry, London, UK.
Rovedatti
Laura
L
Kaur
Rejbinder
R
Spencer
Jonathan P
JP
Brown
Jon T
JT
Morisset
Valerie D
VD
Biancheri
Paolo
P
Leakey
Nicholas A B
NA
Wilde
Jonathan I
JI
Scott
Laurie
L
Corazza
Gino R
GR
Lee
Kevin
K
Sengupta
Neel
N
Knowles
Charles H
CH
Gunthorpe
Martin J
MJ
McLean
Peter G
PG
MacDonald
Thomas T
TT
Kruidenier
Laurens
L
D000328:Adult; D000368:Aged; D000818:Animals; D002121:Calcium Channel Blockers / Q000494:pharmacology; D015220:Calcium Channels / Q000378:metabolism*; D045744:Cell Line, Tumor; D016207:Cytokines / Q000037:antagonists & inhibitors* / Q000096:biosynthesis*; D006801:Humans; D015212:Inflammatory Bowel Diseases / Q000276:immunology* / Q000378:metabolism / Q000473:pathology*; D007413:Intestinal Mucosa / Q000276:immunology / Q000378:metabolism / Q000473:pathology; D019169:Jurkat Cells; D008875:Middle Aged; D009924:Organ Culture Techniques; D018408:Patch-Clamp Techniques; D051381:Rats; D020825:T-Box Domain Proteins / Q000037:antagonists & inhibitors* / Q000502:physiology; D016176:T-Lymphocyte Subsets / Q000276:immunology* / Q000378:metabolism* / Q000473:pathology; D055815:Young Adult; D000098815:T-bet Transcription Factor
D002121:Calcium Channel Blockers; D015220:Calcium Channels; D016207:Cytokines; D020825:T-Box Domain Proteins; D000098815:T-bet Transcription Factor