BMC Genomics BioMed Central Research article Open Access Genomics and proteomics of immune modulatory effects of a butanol fraction of echinacea purpurea in human dendritic cells Chien-Yu Wang1, Vanisree Staniforth1, Ming-Tsang Chiao1, Chia- Chung Hou1, Han-Ming Wu3, Kuo-Chen Yeh1, Chun-Houh Chen3, Pei- Ing Hwang3, Tuan-Nan Wen2, Lie-Fen Shyur*1 and Ning-Sun Yang*1 Address: 1Agricultural Biotechnology Research Center, Academia Sinica, Taipei 115, Taiwan, 2Institute of Plant and Microbial Biology, Academia Sinica, Taipei 115, Taiwan and 3Institute of Statistical Science, Academia Sinica, Taipei 115, Taiwan Email: Chien-Yu Wang - [email protected]; Vanisree Staniforth - [email protected]; Ming-Tsang Chiao - [email protected]; Chia-Chung Hou - [email protected]; Han-Ming Wu - [email protected]; Kuo-Chen Yeh - [email protected]; Chun- Houh Chen - [email protected]; Pei-Ing Hwang - [email protected]; Tuan-Nan Wen - [email protected]; Lie- Fen Shyur* - [email protected]; Ning-Sun Yang* - [email protected] * Corresponding authors Published: 13 October 2008 Received: 1 April 2008 Accepted: 13 October 2008 BMC Genomics 2008, 9:479 doi:10.1186/1471-2164-9-479 This article is available from: http://www.biomedcentral.com/1471-2164/9/479 © 2008 Wang et al; licensee BioMed Central Ltd. This is an Open Access article distributed under the terms of the Creative Commons Attribution License (http://creativecommons.org/licenses/by/2.0), which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited. Abstract Background: Echinacea spp. extracts and the derived phytocompounds have been shown to induce specific immune cell activities and are popularly used as food supplements or nutraceuticals for immuno-modulatory functions. Dendritic cells (DCs), the most potent antigen presenting cells, play an important role in both innate and adaptive immunities. In this study, we investigated the specific and differential gene expression in human immature DCs (iDCs) in response to treatment with a butanol fraction containing defined bioactive phytocompounds extracted from stems and leaves of Echinacea purpurea, that we denoted [BF/S+L/Ep]. Results: Affymetrix DNA microarray results showed significant up regulation of specific genes for cytokines (IL-8, IL-1β, and IL-18) and chemokines (CXCL 2, CCL 5, and CCL 2) within 4 h after [BF/S+L/Ep] treatment of iDCs. Bioinformatics analysis of genes expressed in [BF/S+L/Ep]-treated DCs revealed a key-signaling network involving a number of immune-modulatory molecules leading to the activation of a downstream molecule, adenylate cyclase 8. Proteomic analysis showed increased expression of antioxidant and cytoskeletal proteins after treatment with [BF/S+L/Ep] and cichoric acid. Conclusion: This study provides information on candidate target molecules and molecular signaling mechanisms for future systematic research into the immune-modulatory activities of an important traditional medicinal herb and its derived phytocompounds. Background North American Indians during the 17th century and was Echinacea spp., commonly known as purple coneflower, is later introduced to Europe. Its use became popular again indigenous to North America. The use of Echinacea spp. as in the early 1990s and continues today. Currently, Echina- an herbal remedy originated in the medicinal culture of cea extracts from whole plant or specific tissue (e.g., root Page 1 of 20 (page number not for citation purposes) BMC Genomics 2008, 9:479 http://www.biomedcentral.com/1471-2164/9/479 or aerial parts) are among the top-selling medicinal or partitioned fraction (BF) of the S+L tissue extracts of E. food supplement products in the United States and purpurea [BF/S+L/Ep] and cichoric acid (a major compo- Europe [1-3]. Recent studies have shown that treatment nent of this fraction) through Affymetrix gene chip micro- with specific Echinacea extracts activates macrophages, array analyses. High-resolution 2-D gel electrophoresis, natural killer cells, or other immune cells [4-6]. Echinacea MALDI-TOF mass spectrometry (MS), tandem MS-MS extracts have also been reported to stimulate the secretion analysis, and bio-informatics database systems were sub- of cytokines such as tumor necrosis factor-alpha, inter- sequently employed for proteomics studies in parallel feron, interleukin-1 and interleukin-6 [7-10]. In vivo stud- with the genomics studies. Results from these analyses ies have shown that treatment with Echinacea extracts can and cell-based bioactivity-guide assays suggest that groups increase the number of white blood cells in the circulatory of differentially expressed genes, specific functional genes, system [11], enhance phagocytosis [12], and trigger the and the associated molecular signaling networks can be alternate complementary pathway [13]. Echinacea extracts employed as potential targets for future systematic studies have been marketed as possible immune stimulators or of the response of human DC systems, as a response to tra- enhancers worldwide. These phyto-extracts have been ditional herbal medicine formulations or their derived actively evaluated in various clinical studies as candidate phytocompounds. therapeutics or preventive remedies for upper respiratory tract infections, common cold, urogenital infection and Results wound healing [14-19]. However, the results from various Effect of [BF/S+L/Ep] extract on expression of cell surface studies on the efficacy of Echinacea extracts for prevention markers in human iDCs of experimental colds or common cold have been contro- Flow cytometry revealed that [BF/S+L/Ep] enhanced the versial [16-20]. The most recent study, as a meta-analysis expression of CD83, a key marker for DC maturation in for evaluating the effect of Echinacea, addressed again on iDCs, as compared with vehicle (0.1% DMSO) treatment the potential use and problems of Echinacea as remedy for (Figure 1) and the percentage of CD83+ expressing cells common cold/flu [20,21]. increased, from 20% to 45% with 10, 50 and 100 μg/ml [BF/S+L/Ep]. However, treatment with the ethyl acetate Dendritic cells (DCs) are involved in a spectrum of (EA) fraction of the S+L extract at 1, 10, or 50 μg/ml sig- immune cell functions, including antigen-presentation nificantly decreased the percentage of CD83+ expressing and phagocytic activity, and play important roles in both cells from 20% to 0% (Figure 1). The observed effects were innate and adaptive immunities [22]. DCs can capture not caused by cellular mechanisms related to cytotoxicity, and transfer molecular or cellular information from the since MTT assay results indicated that treatment with EA body's outside or interface environment to cells of differ- ent immune systems. These cells are not only critical for the induction of primary immune responses but are also 60 S+L Crude CD83 involved in the regulation of T cell-mediated immunity S+L EA 50 [22]. Recently, a series of studies developed DC-based S+L BuOH S+L Water immunotherapy or vaccine protocols designed to elicit 40 specific immunity against certain cancers [12,23,24]. Echi- nacea plant extracts have been shown to have immune- 30 modulatory effects [3,11-13], and we recently reported on the possible use of Echinacea purpurea (Ep) phytocom- 20 pounds as immune-modifiers for human DC activity [25]. Relative activity 10 Echinacea extracts have significant and specific modula- (% of positive cells in FACS assay) (% of positive cells tory effects on human DCs, but these effects are plant tis- 0 sue-specific, the bioactivity varying greatly between root 0 20406080100 and shoot plus leaf (S+L) tissues. In this follow-up study, Concentration (μg/ml) we further investigated in detail the effect of a partially purified and chemically defined Ep phytocompound mix- Specific(S+L)cellsFigure (iDC) extracts 1bioactivities of E. purpureaof three insubfractions human immature of stem dendritic and leaf ture on human DCs. Specific bioactivities of three subfractions of stem and leaf (S+L) extracts of E. purpurea in human Recent gene expression profiling in DCs have shown that immature dendritic cells (iDC). iDCs were treated for DCs can actively process environmental signals and acti- 24 h with the S+L tissue extracts and the derived ethyl ace- vate different transcriptional programs in response to dis- tate (EA), butanol (BuOH), or water fractions. Test cells were subsequently analyzed for cell-surface marker CD83 tinct stimuli [26]. In this study, we used functional expression by flow cytometry. genomics to analyze changes in gene expression in human immature DCs in response to treatment with the butanol- Page 2 of 20 (page number not for citation purposes) BMC Genomics 2008, 9:479 http://www.biomedcentral.com/1471-2164/9/479 (1 to 50 μg/ml) or [BF/S+L/Ep] (1 to 100 μg/ml) showed range. The amount of cichoric acid and rutin were deter- 98% to 125% cell viability as compared to vehicle treat- mined as 8.4% (w/w) and 22.3% (w/w), respectively, of ment (data not shown). Thus, the [BF/S+L/Ep] fraction the dry weight of the [BF/S+L/Ep] fraction. Together, the but not the EA fraction could enhance the maturation of results show that this [BF/S+L/Ep] phytocompound mix- iDCs. To reach this conclusion, it is essential for us to rule ture was effectively standardized and defined with a spe- out the possibility that lipopolysaccharide (LPS), as a bac- cific metabolite profile containing several candidate index terial endotoxin contamination of the [BF/S+L/Ep] extract compounds (Figure 2). The standardized [BF/S+L/Ep] preparations, might have contributed to the observed fraction was then systematically used in the following bio- results in the present CD83 assay and the subsequent activity assays to evaluate its immune regulatory effects in functional genomics and proteomics studies.