Schematic showing the cellular pathways by which curcumin (NEC) inhibits macrophage inflammatory signaling.

<p>Curcumin regulates inflammatory pathways at multiple levels: i) downregulation of TLR4 expression on the surface of cells and ii) inhibition of p65 and IκBα phosphorylation in the activation of NFκB. Thus, NEC inhibits transcriptional regulation <i>in vivo</i> of NFκB target genes, including RAGE, MCP-1, and NOS2, which leads to suppressed macrophage-mediated inflammatory responses.</p

Curcumin labels monocytes and inhibits macrophage migration in human cells.

<p><b>A</b>, Human cell lines Jurkat, Clone E6-1 and THP-1 were cultured in complete RPMI medium, supplemented with various amounts of curcumin or vehicle, and analyzed by FACS. Representative results are shown. <b>B</b>, THP-1 (top) and primary human monocyte derived macrophages (bottom) were treated with the indicated concentrations of curcumin and cell viability was measured over time by MTT assay. Values are expressed as fold changes relative to initial baseline levels. <b>C</b>, THP-1 cells were differentiated into macrophages and isolated by adherence to culture plates. Scratch assays were performed on adherent cells with or without curcumin to measure cell migration. Representative images (left) and migrated cell counts (right). <b>D</b>, Primary macrophages were isolated from healthy human blood samples (<i>n</i> = 4) and subjected to migration assays with and without curcumin (20 µM) for the indicated time. Representative images (left) and relative fold changes of migrated cells (right) are shown. Original magnification X 40. *  =  <i>p</i>≤0.05 versus vehicle. n.s.  =  not significant versus vehicle.</p

LPS-induced NFκB reporter gene expression is suppressed with nano-emulsion curcumin (NEC) in mice when compared to equivalent concentration of suspension curcumin (SC).

<p>Transgenic BALB/C-Tg(NFκB-RE-luc)-Xen mice (<i>n</i> = 13) were treated with 1 g/kg NEC, equivalent component concentrations of the nano-emulsion vehicle, or SC by oral gavage 10 min prior to LPS injection (IP, 2 mg/kg), and imaged (IVIS 200; 150 mg luciferin/kg IP) at 0 h, 2 h, 4 h and 6 h. The whole animal, including areas covering the thymus, lymph nodes, and the abdominal region were outlined and subjected to relative photon counting. <b>A,</b> Representative whole body bioluminescent images; <b>B,</b> Digitization of emitted light photons. The fold change in luminescent intensity is expressed relative to the photon counting in the mice at 0 h. Data were analyzed using the mixed effect model, as detailed in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0111559#s2" target="_blank">methods</a>. *  =  <i>p</i><0.0001 versus SC. #  =  <i>p</i><0.0001 versus vehicle. IP  =  intraperitoneal.</p

FACS analyses show that NEC selectively diminishes levels of blood monocytes.

<p>Leukocytes were isolated from whole blood collected from BALB/c mice (<i>n</i> = 6) before and 30 min after oral administration of NEC (1 g/kg). <b>A</b>, FACS analyses of leukocytes measuring the expression of cell surface markers: T-cells (CD3, CD4, or CD8), B-cells (B220), or monocytes (F4/80). * <i>p</i>≤0.05 versus time zero. <b>B</b>, Representative FACS analysis of F4/80⁺ expression.</p

NEC suppresses LPS-induced TLR4 and RAGE expression in addition to blood monocyte accumulation.

<p>BALB/c mice (<i>n</i> = 10) were treated with NEC (1 g/kg) or vehicle by oral gavage and injected with LPS (IP, 2 mg/kg) 10 min afterward. At 4 h, whole blood was collected and leukocytes were isolated for flow cytometry. <b>A</b>, Quantitation (top) and representative images (bottom) from FACS analysis to measure F4/80⁺ cells. <b>B</b>, FACS analysis measuring TLR2, RAGE, or TLR4 expression on the surface of cells. <b>C</b>, ELISA of MCP-1 expression from mouse serum. *  =  <i>p</i>≤0.05 versus vehicle. IP  =  intraperitoneal.</p

Curcumin inhibits mouse macrophage-migration both <i>in vivo</i> and <i>in vitro</i>.

<p>BALB/c mice (<i>n</i> = 6) were oral gavaged daily with vehicle or NEC (1 g/kg/day) for 5 days before intraperitoneal injection of thioglycollate (3 mL, 3% w/v) to induce inflammation. Following 6 days of continued oral administration of NEC or vehicle daily, peritoneal cells were isolated for flow cytometry. <b>A</b>, Representative images following FACS analysis to measure levels of B-cells (B220⁺), T-cells (CD3⁺) or macrophages (F4/80⁺). <b>B</b>, Cell subtype quantitations detected from total peritoneal cell isolates by FACS analysis. <b>C</b>, RAW 264.7 cells were plated to confluence and streaked. Cell migration was observed after 4 d with and without supplementing curcumin (20 µM) to the medium. Representative images taken from duplicated experiments. Original magnification X 80. *  =  <i>p</i>≤0.05 versus vehicle.</p