Yin et al. BMC Genomics 2010, 11:612 http://www.biomedcentral.com/1471-2164/11/612 cell migration/trafficking activities of DCs under ex vivo and in vivo conditions. Since the 1990s, the use of Echinacea spp. as a medic- inal plant or food supplement has gained popularity in the USA and Europe [11,12], and is now even recognized in Asia (e.g., China). The possible effects of Echinacea spp. extracts on in vitro activation of macrophages, nat- ural killer (NK) cells, and other immune cell types [13-15], and on stimulating the expression of cytokines such as tumor necrosis factor-alpha (TNF-a), interferon, interleukin-1 and interleukin-6 [16] have all been reported. In vivo studies showed that treatment with Echinacea extracts can increase white blood cell popula- tions in the circulation system [17], and enhance phago- cytosis [18]. E. purpurea extracts have been evaluated in clinical trials for efficiency against the common cold, but the controversial results are continuously disputed [9,10,12,19,20]. To systemically assess the immunomodu- latory effects on mouse DCs in vivo, we employed a chemically defined Echinacea purpurea extract, termed [BF/S+L/Ep], containing hypoxanthine, chlorogenic acid, caffeic acid, cichoric acid, quercetin-3-O-rhamnosyl- (1®6)-galactoside, kaempferol-3-O-rhamnosyl-(1®6)- galactoside and rutin [10]. Profiling of specific and global gene expression patterns by DNA microarray analysis coupled with proteomic ana- lysis provides a useful approach for the investigation of complex biological phenomena [21,22] as we have pre- viously shown for immune cell systems [9,10]. In this study, we used a network knowledge-based approach to analyze genome-wide transcriptome activity in vitro and in vivo, for correlation to specific proteome activities and special functional phenotypes in mouse immature BMDCs, in response to treatment with [BF/S+L/Ep]. Our findings suggest that [BF/S+L/Ep] was able to modulate cell adhesion-, cell mobility-, cytokine- and NF- B sig- naling-related activities in primary cultures of mouse DCs. In vivo trafficking experiments using ex vivo-treated DCs demonstrated that [BF/S+L/Ep] could enhance the mobility of DCs to target specific lymphoid organs in test mouse. Moreover, bioinformatics studies allowed us to predict several candidate target molecules for the future translational studies of this or other phytocompound mixtures. The significance of our findings and potential application to future studies of human DCs are discussed. Results 1. Expression of DC markers in response to treatment with [BF/S+L/Ep] Initially, we set out to examine whether [BF/S+L/Ep] could influence the maturation activity of BMDCs. Mouse bone marrow cells were isolated and cultured for 9 days in RPMI-1640 supplemented with 200 U/mL GM- CSF. DCs cultured in the presence of GM-CSF showed Page 2 of 18 the functional and phenotypic characteristics of the immature stage and could be further differentiated in vitro into mature DCs. LPS, at 1 μg/mL, was then used to induce maturation of DCs. Some test cells were ana- lyzed for the effect of [BF/S+L/Ep] for 24 h. Since con- centrations of [BF/S+L/Ep] greater than 100 μg/mL were slightly cytotoxic to test dendritic cells (evaluated using MTT cell viability assays, data not shown), [BF/S+L/Ep] was used at a concentration of 75 μg/mL, where no cyto- toxicity was detectable. As seen in Figure 1, sharp peak signals were obtained in flow cytometry analyses, demon- strating that high quality DCs were routinely obtained. Flow cytometry analysis on the expression of CD40, CD80, CD86, MHC class II and CD11c on BMDCs showed that the control group expressed lower levels of co-stimulatory and activation molecules than the LPS- treated groups. There was no significant difference between the DMSO-treated and [BF/S+L/Ep]-treated groups (Figure 1). Compared with the LPS group, BMDCs co-treated with 75 μg/ml [BF/S+L/Ep] and 1 μg/ mL LPS only showed a weak inhibition of CD40 at 24 h post-LPS treatment, whereas expression of CD40, CD86, MHC class II and CD11c were not significantly affected. These data indicate that [BF/S+L/Ep] did not have a sig- nificant effect on the phenotypic maturation of mouse BMDCs. 2. Differential gene expression in immature BMDCs as a response to treatment with [BF/S+L/Ep] We next investigated the expression of specific genes related to the cellular biochemistry and physiology of immature DCs. Affymetrix DNA microarray analyses showed that, although the gene expression of the cell surface markers tested above were not affected, a series of other genes related to DC activities were affected by 75 μg/ml [BF/S+L/Ep]. Initial experimental setups using biological sample replicates showed that our test cell culture systems and experimental protocols, e.g., RNA preparation, were highly reproducible and consistent, e.g., Pearson’s Correlation values for test biological sam- ple replicates were between 0.993 and 0.996 (Figure 2). All genes identified from the raw data of our Affymetrix gene chip analyses were subjected to a gene filtering proctocol, using established statistics software (Spotfire), to select and identify candidate differentially expressed genes for further studies. We chose genes that scored positive in at least 4 different chips and that had a coef- ficient of variation (CV) of over 0.4. A total of 907 and 1256 genes had changed expression significantly by 4 h and by 12 h post-treatment with [BF/S+L/Ep], respec- tively. Among these, 172 and 264 genes respectively were identified as immune-related genes (Table 1). For up-regulated or down-regulated genes, only those genes that showed at least a 2-fold change in RNA transcript