Figure 7

Hemoglobin, a CO scavenger, reduces the anti-inflammatory effects of carnosol and curcumin treatment in LPS-stimulated DC. DC from healthy donors (n = 3) were treated with carnosol (10 µM) or curcumin (10 µM) either alone or in the presence of the CO scavenger hemoglobin (Hb; 10 µM) for 6 hours prior to stimulation with LPS. Cells were stained with fluorochrome conjugated antibodies specific for CD80 and CD86 after 24 hours and analysed by flow cytometry. Pooled data depicting expression of CD80 and CD86 in (A) carnosol and (B) curcumin treated DC. Results shown are mean (±SEM) of the measured MFIs, expressed as percentages of the vehicle controls. (C) Pooled data depicting mean (±SEM) concentration of IL-23 in supernatants of DC treated with carnosol or curcumin, with or without the addition of Hb (means of three technical replicates per donor). (D) DC from healthy donors (n = 4) treated with carnosol (10 µM) or curcumin (10 µM), with or without Hb (10 µM), were incubated with DQ-OVA as described above. Representative dot plots depicting DQ-Ova uptake by DC treated with carnosol, curcumin and Hb are from one donor. (E) Pooled data depicting mean (±SEM) percentage DQ-Ova uptake of DC treated with carnosol and curcumin either alone or in the presence of Hb. (F) Western blot depicting HO-1 expression in DC either untreated (UT) or treated with Hb (10 µM) for 24 hours. Full length blot is presented in Supplemental Fig. 8. Statistical significance was determined by one-way ANOVA, with Dunnett’s multiple comparisons post hoc test to compare treatment groups against the control group (denoted by asterisks) and Sidak’s multiple comparisons post hoc test to compare the means of preselected pairs of groups (denoted by hash marks). (****p < 0.0001, *p < 0.05, ^###p < 0.001, ^##p < 0.01, ^#p < 0.05).