ERK_1/2 involvement in curcumin-mediated up-regulation of late-phase CD69 expression.

<p>(A, B) CD4⁺ T cells were cultured in the presence of anti-CD2/CD3/CD28 antibody-coated beads only (Act.; 1∶10 for bead to cell) or with 2 µg/mL curcumin (Cur.) for 3 days. Cells were then harvested and the percentage of CD69⁺ cells was determined by flow cytometric analysis. (A) After 48 hours of culture, cells were treated with an additional 2 µg/mL of curcumin (Cur. 48 hours). The number in each panel indicates the total percentage of CD69⁺ cells. The results are the representative of 3 replicate experiments yielding similar results. (B) After 48 hours of culture, cells were treated with 10 µM of U0126 (ERK inhibitor), SP600125 (JNK inhibitor), or SB203580 (p38 MAPK inhibitor). (C) CD4⁺ T cells were cultured in the presence of anti-CD2/CD3/CD28 antibody-coated beads (Act.; 1∶10 for bead-to-cell ratio) for 3 days, with 10 µM of U0126 (ERK inhibitor), SP600125 (JNK inhibitor), or SB203580 (p38 MAPK inhibitor) added 1 hour prior to treatment with an additional 2 µg/mL of curcumin (Cur. 48hrs) after 48 hours of culture. CD69 expression was assessed by flow cytometry. Data are presented as the mean ± SD. **<i>P</i><0.01, ***<i>P</i><0.001.</p

Curcumin inhibits CD4⁺ T cell expansion induced by CD2/CD3/CD28 signaling without inducing cell death.

<p>CD4⁺ T cells were cultured in the presence of anti-CD2/CD3/CD28 antibody-coated beads only (Act.) or with either 0.2 or 2 µg/mL of curcumin (Cur.) for the indicated time periods. Act. (1/2) and Act. (1/10) represent bead-to-cell ratios of 1∶2 and 1∶10, respectively. (A, B) For cell proliferation assay, the cells were labeled with CFSE prior to culture, and harvested at 3 days of culture. (A) Cell generations (G0∼G4) were calculated and (B) plotted as the percentage of total cells by using flow cytometry and FlowJo software. (C) Results of an MTT cell proliferation assay to assess cell numbers at 1, 2 and 3 days of culture. (D) Cells were harvested at the indicated time points and labeled with an anti-Annexin V antibody and PI. The numbers in the plot indicate the percentage of cells in the respective areas. Data are representative of 3 experiments yielding similar results. (C and D) Curcumin was added at a concentration of 2 µg/mL. (B and C) Data are presented as the mean ± SD. *<i>P</i><0.05, **<i>P</i><0.01, ***<i>P</i><0.001.</p

TGF-β1 is associated with curcumin-mediated generation of regulatory T cells at late phase.

<p>CD4⁺ T cells were cultured in the presence of anti-CD2/CD3/CD28 antibody-coated beads only (1∶10 for bead-to-cell ratio) or with 2 µg/mL of curcumin (Cur.) for 3 days, and then transferred to a new cell culture plate and incubated with a fresh media for an additional 3 days. (A) Cells were collected at the indicated time points and labeled with anti-CD25 and anti-Foxp3 antibodies. The cells were then washed and analyzed for CD25 and Foxp3 expression by flow cytometry. The numbers in each panel and the numbers in blankets indicate the percentage of CD25^hiFoxp3⁺ cells in total and among CD25⁺ cells, respectively. (B) After 6 days of culture, cells were collected and percentage of CD25^hiFoxp3⁺ cells in total and among CD25⁺cells was determined by flow cytometric analysis. The empty and filled bars indicate cells treated with beads only and cells treated with both beads and curcumin, respectively. (C) A TGF-βRI kinase inhibitor (5 µg/mL) was added after 3 days of culture. The percentage of Foxp3⁺ cells was determined by flow cytometry. (D) After 3 days of culture, the cells were washed with PBS and then co-cultured with CFSE-labeled autologous CD4⁺ T cells with or without CD2/CD3/CD28 stimulation for 3 days. The cell proliferation was determined by flow cytometric analysis. (B, C and D) Data are presented as the mean ± SD. *<i>P</i><0.05, **<i>P</i><0.01, ***<i>P</i><0.001.</p

Curcumin attenuates late-phase CD69 decline and up-regulates late-phase CCR7 and L-selectin expression.

<p>CD4⁺ T cells were cultured in the presence of anti-CD2/CD3/CD28 antibody-coated beads only (Act.) or with 2 µg/mL curcumin (Cur.) for 3 days. Act. (1/2) and Act. (1/10) indicate a 1∶2 and 1∶10 bead-to-cell ratio, respectively. (A) Cells were harvested at the indicated time point and the percentage of CD69⁺ cells was determined by flow cytometric analysis. (B–D) After 3 days of culture, cells were harvested, washed with PBS, and then transferred to a new cell culture plate in fresh media for an additional 3 days. The cells were then stained and analyzed by flow cytometry. (B) Histograms of CD69 expression and the total percentage of CD69⁺ cells. (C) The numbers in each plot and the number in blanket indicate the percentage of cells in each respective area and the percentage of CD69⁺ cells among cells positive with the Y axis, respectively. (D) The percentage of total cells positive for each molecule. Data are representative of 3 replicate experiments yielding similar results. (A, B and D) Data are presented as the mean ± SD. *<i>P</i><0.05, ***<i>P</i><0.001.</p

Curcumin suppresses CD4⁺ T cell activation and differentiation.

<p>CD4⁺ T cells were cultured in the presence of anti-CD2/CD3/CD28 antibody-coated beads alone (Act.) or with 2 µg/mL curcumin (Cur.) for 3 days. Act. (1/2) and Act. (1/10) indicate a 1∶2 and 1∶10 bead-to-cell ratio, respectively. Cells were harvested at the indicated time points and the expression of (A) CD25 and (B) CD45RO, IL-12Rβ1, CD27, CD40L, CCR7, L-selectin and Integrin β7 was determined by flow cytometric analysis. (A) Mean fluorescence index (MFI) and (B) percentage of the cells expressing each molecule was calculated by using FlowJo software. (C) The supernatants were collected at 3 days of culture and the total cytokine level was determined using sandwich ELISA. (D) After 3 days of culture, cells were collected, extensively washed with PBS, and then transferred to new cell culture plate in fresh media for an additional 3 days. Following culture, the cells were stimulated with PMA and ionomycin plus Brefeldin A for an additional 5 hours, and then intracellular cytokine content was evaluated. The numbers in plots indicate the percentage of cytokine-expressing cells. Results are representative of 3 replicate experiments yielding similar results. (A, B and C) Data are presented as the mean ± SD. *<i>P</i><0.05, **<i>P</i><0.01, ***<i>P</i><0.001.</p

The influence of TGF-β1 on the regulation of CD4⁺ T cell activation following curcumin treatment.

<p>CD4⁺ T cells were cultured in the presence of anti-CD2/CD3/CD28 antibody-coated beads only (Act.; 1∶10 for bead-to-cell ratio) or with 2 µg/mL of curcumin (Cur.) for 3 days. (A) Cell culture supernatants were collected at the indicated time point and total levels of active TGF-β were determined using an ELISA. (B) After 3 days of culture, cells were collected, washed with PBS and then transferred to a new cell culture plate in order to re-culture the cells in fresh media with or without a TGF-βRI kinase inhibitor (5 µg/mL) for an additional 3 days. The percentage of CD25⁺, IFN-γ⁺, IL-10⁺, IL-13⁺ and IL-17⁺ cells was determined by flow cytometric analysis. For detection of cytokine-producing cells, the cells were re-stimulated with PMA and ionomycin plus Brefeldin A for 5 hours. Data are presented as the mean ± SD. *<i>P</i><0.05, **<i>P</i><0.01, ***<i>P</i><0.001.</p