Figure 7
Systemic CRAC channel inhibition alleviates colon inflammation in mice
A–C Acute administration of the CRAC channel blocker CM4620 inhibits CRAC currents (Icrac). (A) Time course of peak amplitudes of ICRAC before and after application of 3 μM CM4620 to HEK293 cells transfected with ORAI1 and STIM1. (B) Current–voltage (I–V) relationship of ICRAC before and after CM4620 treatment. I–Vs were obtained at the time points indicated by the arrowheads in (A). (C) Fractional blockade of ICRAC by CM4620 was measured by comparing current amplitudes before and after CM4620 administration (arrowheads) using the formula 1 − (ICM4620/ICtrl). Data are from eight cells per conditions and shown as the mean ± SEM.
D IBD was induced by i.v. injection of 5 × 105 naïve CD4+ T cells into Rag1−/− host mice followed by oral gavage of mice with 20 mg/kg CM4620 or vehicle (SDD) every other day from days 18–49.
E Relative weight loss of mice treated with vehicle or CM4620; the start of treatment is indicated by the white arrow. Data are the mean ± SEM of nine mice per cohort, statistical analysis by unpaired Student's t‐test, *P < 0.05.
F Concentrations of CM4620 in the serum of mice at day 49, data are the mean ± SEM. Statistical analysis by unpaired Student's t‐test, ***P < 0.001.
G Representative H&E staining of the distal colon of Rag1−/− mice treated with vehicle or CM4620. Colitis scores of nine mice per cohort. Each symbol represents one mouse. Statistical analysis by Mann–Whitney U test Data are the mean ± SEM, ***P < 0.001.
H Number of CD11b+Gr‐1+ neutrophils and CD4+ T cells (normalized to grams of tissue) in the colon lamina propria (CLP) of mice on day 49 quantified by flow cytometry. Each dot represents one mouse. Data are the mean ± SEM of nine mice per cohort.
I Frequencies of RORγt+ Th17 cells and Foxp3+ Treg cells in the CLP of mice treated with CM4620 or vehicle. Each dot represents one mouse. Data are the mean ± SEM of six control and five CM4620 treated mice.
J Frequencies of IFN‐γ, TNF‐α, and IL‐2 producing CD4+ T cells isolated from the CLP, restimulated ex vivo with PMA and ionomycin for 4 h (without addition of CM4620 during stimulation in vitro) and analyzed by flow cytometry. Bar graphs represent the percentages of IFN‐γ+, TNF‐α+, IL‐2+ cells in CD4+ cells (top row) and mean fluorescent intensity (MFI) of IFN‐γ, TNF‐α, IL‐2 on CD4+ cells (bottom row). Each dot represents one mouse. Data are the mean ± SEM of six control and five CM4620 treated mice.
Data information: Statistical analyses in panels (H–J) by an unpaired Student's t‐test: ***P < 0.001 **P < 0.01 *P < 0.05.
Source data are available online for this figure.