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 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

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

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

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

Antituberculosis Activity of a Naturally Occurring Flavonoid, Isorhamnetin

Isorhamnetin (<b>1</b>) is a naturally occurring flavonoid having anticancer and anti-inflammatory properties. The present study demonstrated that <b>1</b> had antimycobacterial effects on <i>Mycobacterium tuberculosis</i> H₃₇Rv, multi-drug- and extensively drug-resistant clinical isolates with minimum inhibitory concentrations of 158 and 316 μM, respectively. Mycobacteria mainly affect the lungs, causing an intense local inflammatory response that is critical to the pathogenesis of tuberculosis. We investigated the effects of <b>1</b> on interferon (IFN)-γ-stimulated human lung fibroblast MRC-5 cells. Isorhamnetin suppressed the release of tumor necrosis factor (TNF)-α and interleukin (IL)-12. A nontoxic dose of <b>1</b> reduced mRNA expression of TNF-α, IL-1β, IL-6, IL-12, and matrix metalloproteinase-1 in IFN-γ-stimulated cells. Isorhamnetin inhibited IFN-γ-mediated stimulation of extracellular signal-regulated kinase and p38 mitogen-activated protein kinase and showed high-affinity binding to these kinases (binding constants: 4.46 × 10⁶ M^–1 and 7.6 × 10⁶ M^–1, respectively). The 4′-hydroxy group and the 3′-methoxy group of the B-ring and the 5-hydroxy group of the A-ring of <b>1</b> play key roles in these binding interactions. A mouse <i>in vivo</i> study of lipopolysaccharide-induced lung inflammation revealed that a nontoxic dose of <b>1</b> reduced the levels of IL-1β, IL-6, IL-12, and INF-γ in lung tissue. These data provide the first evidence that <b>1</b> could be developed as a potent antituberculosis drug

Tamarixetin Exhibits Anti-inflammatory Activity and Prevents Bacterial Sepsis by Increasing IL-10 Production

Sepsis is a systemic inflammatory response to pathogenic infection that currently has no specific pharmaceutical interventions. Instead, antibiotics administration is considered the best available option, despite increasing drug resistance. Alternative strategies are therefore urgently required to prevent sepsis and strengthen the host immune system. One such option is tamarixetin (4′-<i>O</i>-methylquercetin), a naturally occurring flavonoid derivative of quercetin that protects against inflammation. The purpose of this study was to determine whether the anti-inflammatory effects of tamarixetin protect against the specific inflammatory conditions induced in lipopolysaccharide (LPS) or <i>Escherichia coli</i> K1 models of sepsis. Our study showed that tamarixetin reduced the secretion of various inflammatory cytokines by dendritic cells after activation with LPS. It also promoted the secretion of the anti-inflammatory cytokine interleukin (IL)-10 and specifically increased the population of IL-10-secreting immune cells in LPS-activated splenocytes. Tamarixetin showed general anti-inflammatory effects in mouse models of bacterial sepsis and decreased bacteria abundance and endotoxin levels. We therefore conclude that tamarixetin has superior anti-inflammatory properties than quercetin during bacterial sepsis. This effect is associated with an increased population of IL-10-secreting immune cells and suggests that tamarixetin could serve as a specific pharmaceutical option to prevent bacterial sepsis

A Potential Protein Adjuvant Derived from <i>Mycobacterium tuberculosis</i> Rv0652 Enhances Dendritic Cells-Based Tumor Immunotherapy

<div><p>A key factor in dendritic cell (DC)-based tumor immunotherapy is the identification of an immunoadjuvant capable of inducing DC maturation to enhance cellular immunity. The efficacy of a 50S ribosomal protein L7/L12 (rplL) from <i>Mycobacterium tuberculosis</i> Rv0652, as an immunoadjuvant for DC-based tumor immunotherapy, and its capacity for inducing DC maturation was investigated. In this study, we showed that Rv0652 is recognized by Toll-like receptor 4 (TLR4) to induce DC maturation, and pro-inflammatory cytokine production (TNF-alpha, IL-1beta, and IL-6) that is partially modulated by both MyD88 and TRIF signaling pathways. Rv0652-activated DCs could activate naïve T cells, effectively polarize CD4⁺ and CD8⁺ T cells to secrete IFN-gamma, and induce T cell-mediated-cytotoxicity. Immunization of mice with Rv0652-stimulated ovalbumin (OVA)-pulsed DCs resulted in induction of a potent OVA-specific CD8⁺ T cell response, slowed tumor growth, and promoted long-term survival in a murine OVA-expressing E.G7 thymoma model. These findings suggest that Rv0652 enhances the polarization of T effector cells toward a Th1 phenotype through DC maturation, and that Rv0652 may be an effective adjuvant for enhancing the therapeutic response to DC-based tumor immunotherapy.</p></div

Rv0652-treated DCs induce proliferation of CD4⁺ and CD8⁺ T cells and a Th1 response.

<p>Transgenic OVA-specific CD8⁺ T cells (A-i) and OVA-specific CD4⁺ T cells (B-i) were isolated and stained using CFSE. These cells were then co-cultured for 96 h with untreated DCs, DCs pulsed with OVA h with untreated DCs, DCs pulsed with OVA_257–264, Rv0652 (1 ug/ml)-treated DCs pulsed with OVA_257–264, or LPS (200 ng ng/ml)-treated DCs pulsed with OVA_257–264. T-cell proliferation was assessed using flow cytometry and the percentage of proliferating cells is shown in each panel. Bar graphs show the percentage of OVA-specific CD8⁺ T cells (A-ii) and OVA-specific CD4⁺ T cells (B-ii) from 3 independent experiments. ***<i>P</i><0.001 compared to T cells/OVA pulsed-DCs. (C) IFN-gamma production was measured in each culture supernatant from Fig. 5A and 5B after 24 h of culture by performing an ELISA. Data are the mean and SEM of 3 experiments. ***<i>P</i><0.001 compared to the value from T cell/OVA peptide-pulsed DCs.</p