Curcumin alters positive and negative cell surface molecule expression on human monocyte-derived dendritic cells (hu-Mo-DC). Hu-Mo-DC were generated as described in Materials and methods, exposed to curcumin (or complete medium) and matured with lipopolysaccharide (LPS) (where applicable) for 24 h. Flow cytometry was used to compare the phenotypic profile of immature DC (immDC, no LPS), mature DC (matDC, exposed to LPS) and curcumin DC (CurcDC) (exposed to LPS). Data are shown gated to the DC population by forward-/side-scatter and the line (P2) indicates isotype control. (a) CurcDC failed to up-regulate positive co-stimulatory molecules when exposed subsequently to LPS, particularly CD83. (b) Curcumin inhibited expression of negative regulatory molecules PDL1 and PDL2 compared to both matDC and immDC. Immunoglobulin-like transcript 2 (ILT2) and ILT4 expression remained unchanged. These figures are from an individual experiment representative of four performed.

Curcumin generates maturation-arrested dendritic cells (DC). Mature DC (matDC) and curcumin DC (CurcDC) were washed extensively and exposed to additional CD40L and interferon (IFN)-γ to mimic inflammatory conditions in vivo, as might occur following exposure to alloantigen. (a) Compared to matDC, CurcDC demonstrated down-regulated RelB mRNA expression (decreased by 56%, *P < 0·001) consistent with a maturation-arrested state. (b,c) MatDC (b) or CurcDC (c) were fixed and analysed by confocal microscopy. Immunofluorescent staining for nuclear factor (NF)-κBp50 subunit demonstrated failure of up-regulation and nuclear translocation in CurcDC despite a robust maturation stimulus. Cell nuclei [4′6-diamino-2-phenylindole (DAPI)] are coloured blue and NF-kBp50 is coloured green. (d,e) CurcDC demonstrated lower mRNA expression of interleukin (IL)-12p40 (decreased by 63%, *P < 0·001) compared to matDC (d), and failed to produce detectable quantities of IL-12p70 (e). (f) CurcDC were capable of producing significant IL-10, albeit in reduced quantities compared to matDC (*P < 0·001). Reverse transcription–polymerase chain reaction results are mean ± standard deviation of quadruplicate measurements, normalized to a housekeeping gene (HPRT1), and are representative of six independent experiments. Cytokine protein production is from triplicate wells assessed by enzyme-linked immunosorbent assay, and representative of four independent experiments.

Curcumin dendritic cells (CurcDC) demonstrate impaired allostimulatory capacity mediated by CD4⁺CD25^hiforkhead box P3 (FoxP3)⁺ regulatory T cells. (a) T cells were co-cultured with immature DC (immDC) mature DC (matDC) or CurcDC at differing T cell : DC ratios for 5 days and proliferation was determined using [³H]-thymidine incorporation added in the final 18 h of culture. Compared to matDC, CurcDC showed lower T cell allostimulatory capacity at all T cell : DC ratios, comparable to immDC. Results shown are mean ± standard error of the mean (*P < 0·01 matDC versus CurcDC and matDC versus immDC). Results are from an individual experiment and representative of four independent experiments performed. T cells co-cultured in a primary mixed lymphocyte reaction (MLR) were then isolated using CD3⁺ magnetic beads and analysed by flow cytometry. (b) T cells co-cultured with CurcDC demonstrated a 50% reduction in intracellular interferon (IFN)-γ expression compared to matDC. (c) T cell hyporesponsiveness was mediated by the generation of CD4⁺CD25^hiFoxP3⁺ T cells. This population was also CD127^loCD62L⁺, further confirming a regulatory phenotype. (d) Co-culture with CurcDC [and subsequent stimulation with staphylococcal enterotoxin B (SEB)] failed to induce T helper type 17 (Th17) cells; (b–d) demonstrate results from one of five independent experiments.

Curcumin dendritic cells (CurcDC) are tolerogenic. (a) T cells were co-cultured initially with CurcDC or mature DC (matDC), isolated from the stimulating cell population using CD3⁺ magnetic beads, and restimulated in a 5-day mixed leucocyte reaction (MLR) (*P < 0·01). Stimulating DC were harvested from the same donor or third party and subsequent T cell hyporesponsiveness was not limited to the primary donor antigen. (b) Compared to matDC, restimulation of T cells co-cultured initially with CurcDC significantly expanded forkhead box P3 (FoxP3)⁺ regulatory T cells that were CD127^lo. (c) Fluorescence activated sorted CD4⁺ T cells were allostimulated for 5 days in the presence of CurcDC, purified using CD3⁺ immunomagnetic beads and co-cultured with naive syngeneic T cells at ratios of 1:1 to 20:1. Suppression of naive T cell proliferation confirmed regulatory function. These figures are representative of three independent experiments.

Murine curcumin dendritic cells (CurcDC) distribute systemically, promote forkhead box P3 (FoxP3)⁺ regulatory T cell expansion in vivo and impair subsequent alloproliferative responses in a non-alloantigen-specific manner. Splenic CD11c⁺ DC from C57BL/6 (H-2^b) mice were isolated using immunomagnetic beads, stained with 1,1′-dioctadecyl-3,3,3′,3′-tetramethylindodicarbocyanine perchlorate (DiI) and injected intravenously into BALB/c (H-2^d) mice to assess systemic distribution. Seven days after injection, organs were removed, snap-frozen, stained and analysed by confocal microscopy (original magnification, 40×). (a) Spleen from BALB/c mice injected with DiI-labelled DC demonstrated red fluorescent cells (DiI⁺ DC) with co-localization of staining for CD11c (green) and DEC205 (yellow). (b) CD11c⁺ C57BL/6 DC were incubated with 25 µM (CurcDC) or complete medium (immDC) overnight, washed and injected into BALB/c mice. Splenocytes were isolated after 7 days and flow cytometry performed to assess FoxP3 expression. Infusion of immDC or CurcDC both demonstrated an increase in the CD4⁺CD25^hiFoxP3⁺ population compared to naive mice [injected with phosphate-buffered saline (PBS)]. (c) Splenocytes were restimulated ex vivo for 5 days with primary donor (C57BL/6) or third-party donor (C3H) antigen (irradiated mononuclear cells). Infusion of immDC led to an antigen-specific primed immune response. However, CurcDC impaired alloproliferation non-specifically compared to both naive (PBS-injected) and immDC-injected mice (*P < 0·01). Results are expressed as mean ± standard deviation of quintuplicate measurements from one experiment, and are representative of four independent experiments. (d) The responding CD4⁺ T cell population in the secondary mixed lymphocyte reaction was analysed, demonstrating expansion of the CD4⁺CD25^hiFoxP3 following infusion of both immDC and CurcDC, although the increase was substantially greater in the latter. Results are representative of two separate experiments.