Anti-Inflammatory and Immunostimulatory Effects of Extract from Culture of Effective Microorganisms ( ECEM ) Revealed by Functional Genomics and Metabolome Analyses

The extract from culture of effective microorganisms (ECEM) is a secondary metabolite produced in symbiotic culture of a mixture of yeasts and lactic bacteria followed by photosynthetic bacteria. Metabolomics studies have demonstrated that ECEM contained several active ingredients associated with anti-inflammation through interactions with nuclear factor-κB. Functional genomics using DNA microarrays revealed that ECEM had distinctive anti-inflammatory and immunostimulatory actions in RAW264.7 cells. ECEM inhibited the LPS-upregulated expression of one group of pro-inflammatory genes (iNos, Il6 and Cox2) but not another group genes (Tnf, Ccl7 and Dusp2); it also upregulated the expression of Cd1d1, which participates in antigen presentation on lipids, resulting in activation of natural killer T (NKT) cells related to infection defense and cancer. Bacterial glycosylated lipid and other related complex lipids in ECEM are thought to trigger this upregulation of Cd1d1 expression. These results suggest that ECEM functions in the immune response in both inflammation and antigen presentation and that it contains components important for these functions. These novel effects of ECEM could be invaluable for developing functional drinks or health beverages.


Introduction
Extract from culture of effective microorganisms (ECEM) is a secondary metabolite produced by the symbiosis culture of 5 microorganisms: 2 strains of yeasts (Saccharomyces cerevisiae and Candida ethanolica), 2 strains of lactic acid bacteria (Lactobacillus casei and Lactobacillus farraginis), and photosynthetic bacteria (Rhodopseudomonas palustris).The secondary metabolites from a few microorganisms are known to contain invaluable effective compounds, not only for developing drugs to treat cancer or infectious diseases [1]- [3], but also for exploiting functional food and/or drink for health control.Establishing the health claims of ECEM as functional drink requires in-depth analysis because the components and functions of the symbiosis culture metabolite are very complicated and difficult to identify.Functional genomics studies using DNA microarrays are powerful tools for elucidating the biological effects of functional foods and/or drinks containing complex components produced by microorganism culture [4] [5].Metabolome analysis provides high-throughput information regarding the chemical nature of low-molecular weight compounds contained in the complicated secondary metabolites [6] [7].
The first stage of studying the biological effects of newly developed foods or drinks should be performed on an in vitro cellular level rather than that in vivo in animals because of the well-controlled conditions and welldefined results.RAW264.7 cells, a murine macrophage cell line with the potential for antigen presentation, are widely used for in vitro first-stage studies examining the biological effects of functional food and/or drink for health control, as murine model are commonly used in vivo for second-stage studies [8]- [10].
In the current study, we examined the in vitro anti-inflammatory and immunostimulatory functions of ECEM in RAW264.7 cells using genome informatics of DNA microarray analysis.We also identified anti-inflammatory components in ECEM through bioinformatics analysis of metabolome data.Our results demonstrate that ECEM has remarkable health effects for modulating inflammation and immune responses and can be included in functional drinks.

Preparation of the Symbiosis Culture Product, "ECEM"
Two strains of Lactobacillus (L.casei and L. farraginis), two strains of yeast (S. cerevisiae and C. ethanolica), and photosynthetic bacteria (R. palustris) were kind gift from Dr. Teruo Higa (director of the International EM Technology Research Center, Meio University).The first step of symbiosis culture, which included L. casei, L. farragini, S. cerevisiae, and C. ethanolica, was performed in medium containing 7% (v/v) sugarcane molasses for 4 weeks at 30˚C until the solution reached pH 3.5 or less, followed by the addition of new sugarcane molasses medium for an additional 2 -4 weeks at 30˚C; culture was terminated when the pH was 3.5 or less.The obtained symbiosis culture product was adjusted to pH 6.5 -7.5 by adding natural calcium powder.This mixture was used for second-step culture that included R. palustris, which was previously grown in medium containing 0.5% (v/v) yeast extract.The second step of symbiosis culture was carried out in medium composed of the original symbiosis culture product, the R. palustris culture product, and water at pH 7.0 -9.0 for 3 months at 25˚C.The supernatant of the second symbiosis culture product was transferred to another container, followed by maturation for 7 days at 25˚C to obtain final secondary metabolites.The resulting symbiosis culture product was filtered through a 50-nm filter and referred to as ECEM, which was used for subsequent experiments.

Cell Culture and ECEM Treatment
Mouse macrophage like cell line, RAW264.7 cells (Summit Pharmaceuticals International Corporation, Osaka, Japan), was cultured with RPMI 1640 medium containing 10% fetal calf serum in 5% CO 2 at 37˚C.To examine the in vitro function of ECEM, the cells were incubated with the culture medium containing the indicated concentrations of ECEM or LPS from Escherichia coli O55 (Wako Pure Chemical Industries, Ltd., Osaka, Japan) for 24 h.

Cell Viability Assay
A colorimetric assay for cell viability was performed using a WST-1 reagent Kit (Takara Bio Inc., Shiga, Japan).Cells were incubated with the culture medium for 24 at 37˚C and 5% CO 2 followed by addition of 10 μL of the WST-1 reagent.After incubation for 4 h at 37˚C and 5% CO 2 , absorbance at 450 nm was measured using Bio-Rad Microplate Reader (iMark 168 -1130 JA; Bio-Rad Laboratories, Inc., Hercules, CA, USA).Cell culture examinations were performed under the conditions for cell viability with more than 80%.

Metabolome Analysis
For metabolome analysis, 10 mL ECEM was concentrated by lyophilization followed by rehydration in 1 mL water.The concentrated ECEM was centrifuged at 15,000 rpm for 1 min at 25˚C to remove water-insoluble material(s) and then filtered with 10-K cut-off membrane.The obtained water-soluble and low-molecular weight fraction of ECEM was subjected to metabolome analysis.Metabolome analysis was performed using an LC-TOF-MS system with a combination of Waters LCT Premier XE/ACQUITY UPLC and ACQUITY UPLC HSS T3 column using an elution solvent with a liner gradient of 0.1% formic acid and acetonitrile.From the detected peak signal, the corresponding chemical formula, molecular mass, and/or compound name were deduced using a data mining system of metaATOne (mata Profiling and meta Compareing).

RNA Preparations and RIN Analysis
RNA preparation was performed as described previously [11].Briefly, total RNA was extracted from the harvested RAW264.7 cells using Isogen (NipponGene Co., Ltd., Tokyo, Japan).Aqueous phase samples were DNase-treated using the RNase-Free DNase Set (QIAGEN, Inc., Hilden, Germany).The extracted RNA was further purified using an RNeasy MinElute Cleanup Kit (QIAGEN), and RNA quantity and purity were evaluated photometrically at 260 nm, 280 nm, and 320 nm using an Ultrospec 2000 (GE Healthcare Bio-Sciences AB, Little Chalfont, UK).RNA quality was assessed using an Agilent 2100 Bioanalyzer (Agilent Technologies, Inc., Santa Clara, CA, USA) and RNA Integrity Number (RIN) was calculated.RNA samples with RIN above 7 were used for gene expression analyses using DNA microarray and real-time reverse transcription (RT)-PCR.

DNA Microarray Analysis
Total RNA (200 ng) from the RAW 264.7 cells was used to generate cDNA and Cy3-labeled cRNA with a Low Input Quick-Amp Labeling Kit (Agilent Technologies, Inc., Santa Clara) according to the manufacturer's instructions.The labeled cRNA was photometrically examined to determine the quantity and dye-incorporation ratio with an Ultrospec 2000, and it was hybridized to a Mouse Gene Expression 4 × 44 K v2 Microarray (Agilent Technologies, Inc.).The array was scanned with GenePix 6000B (Molecular Devices, LLC, Sunnyvale), and the obtained image was processed with GenePix Pro 6.0 Software (Molecular Devices, LLC).The features were manually examined, and spots of poor quality were flagged and filtered out.
The signal data for the features (spots of array) were then imported into GeneSpring 12.6 (Agilent Technologies), and further analyses were performed using the software.The signal data from the arrays were normalized using the 75th percentile method [12], and baseline transformation was performed with the median of the control samples.Quality control was performed in order to filter out signal data with standard errors over 0.2.
To analyze the differentially expressed genes, the fold change vs. the control samples was calculated, and genes with a fold change greater than 2.0 or less than 0.5 were extracted for the ECEM-treated groups.Gene ontology analysis of the selected genes was performed using GeneSpring 12.6 software, and gene sets with a corrected p value < 0.1 were considered significant.

LPS Assay
ECEM contained 11.3 ng/mL LPS.This appeared to be derived from the gram-negative photosynthetic bacteria (R. palustris).

Metabolome Analysis
Metabolome analysis using the LC-TOF-MS system provided useful information regarding the effective components in ECEM.There were 84 major ingredient peaks with intensities higher than 1000 in the water-soluble and low-molecular weight fractions of ECEM.Thirty-four peaks of the 84 major peaks were assigned as known compounds.The biological functions of these known compounds were deduced through bioinformatics analysis using PubMed and other biomedical databases.Table 1 shows the compounds found in the ECEM and their biological roles associated with estrogen receptor (16-alpha-hydroxyestrone), anti-inflammation (embelin, gingerol, and diallyl sulfide), and antimicrobials or antibiotics (4-amino-4-deoxychorismate and phytuberin).

Functional Genomics
Since ECEM contained LPS, gene expression events occurred in RAW cells during incubation with ECEM, which were thought to have resulted from three factors: LPS-mediated alteration in gene expression, ECEM-alleviation to the LPS-mediated alteration, or ECEM-specific and LPS-independent gene expression.We conducted functional genomics analysis using DNA microarray for the RAW cell lysate; cells were obtained by culture for 24 h with medium alone (control), 10 ng/mL LPS, and 10% ECEM.This enabled identification of the possible functions of ECEM related to genomic event(s) in ECEM-treated RAW cells; these included 1) alleviative or 2) no alleviative effect on LPS-mediated alteration in gene expression and/or 3) LPS-independent upregulation or downregulation in gene expression.
Table 4 shows two genes, Cd1d1 and Abcb9, which were upregulated specifically by ECEM but not LPS.These genes are known to be associated with antigen presentation in the immune response.Cd1d1 recognizes lipid antigens and activates NKT.This suggests that ECEM contains simple and/or complex lipid compound(s) from the cell wall of both bacteria and yeast.Little upregulation of Cd1d1 was observed in RAW cells stimulated with 10 ng/mL LPS though LPS was aglycosylated lipid and had lipid-A moiety.
As shown in Table 5, five genes, Bnip3, Ddit4, Ero1l, Hipk2, and Ier3, were downregulated specifically by ECEM but not LPS.These genes were categorized as apoptosis-related by ontology analysis using Gene Spring, suggesting a regulatory effect of ECEM on apoptosis.

Discussion
ECEM is a secondary metabolite produced in symbiosis culture with 5 microorganisms, including 2 strains of yeasts, 2 strains of lactic acid bacteria, and photosynthetic bacteria.Symbiosis culture involves co-cultivation of 2 or more different microorganisms.Symbiosis culture mimics some ecological situations where microorganisms co-exist within complex microbial communities.This mixed culture leads to significantly enhanced production of constitutively present compounds and/or to accumulation of cryptic compounds that are not detected in axenic cultures [25] Symbiosis culture can result in increased yields of previously undetected metabolites and, importantly, the induction of previously unexpressed pathways for bioactive constituents [6] [26].These secondary induced products that are yielded through co-cultivation of mixed microorganisms are known to be valuable natural metabolites [1].Numerous secondary metabolites are defense compounds for plant that protect against biotic and abiotic stresses such as infection, predation, ultraviolet radiation, and drought [2].Other secondary metabolites act as regulatory molecules and exhibit powerful bioactive properties in animals, including humans, by acting as antioxidants, anti-inflammatory, and immune-enhancing agents, as well as antimicrobials [3].In humans, some microorganisms also synthesize their own secondary metabolites that regulate patho-physiological functions such as inflammation and immune responses [27].
Because of the complexity of secondary metabolites, advanced and high-throughput analytical methods (e.g., metabolomics and functional genomics) are important for identifying effective constituents and the biological functions of these metabolites.In the current study, we conducted both metabolomics analysis, using LC-TOF-MS, and functional genomics analysis, using DNA microarray, to investigate the constituent-function relationship of ECEM.We employed a murine macrophage cell line, RAW264.7 cells, for in vitro study because RAWcells are widely used to investigate the biological effect of natural products, including secondary metabolites [8] and we have a plan to perform second stage in vivo study using mouse model.
LPS is a potent mediator in gram negative bacteria and elicits inflammatory and/or immune responses by altering the expression of genes, including iNos, Il6, and Tnf, which are associated with these responses [28].Previous studies have examined upregulated expression of these pro-inflammatory genes in LPS-stimulated RAW 264.7 cells [29].Since ECEM contained 11.3 ng/mL LPS, one can consider two events may have occurred: a synergistic or no effect by ECEM on LPS action upon co-culture of RAW cells with ECEM or LPS.Interestingly, however, DNA microarray showed that ECEM inhibited LPS-upregulated expression of genes, including iNos and Il6, which are related to the inflammatory response (Table 2).The inhibitory effect of ECEM was confirmed by co-culture of RAW cells with both ECEM and LPS (Figure 1(a) and Figure 1(b)).Unexpectedly, ECEM exhibited little inhibitory action on LPS-upregulated expression of some pro-inflammatory genes, including Tnf (Table 3 and Figure 1(c)).This characteristic finding suggests that ECEM has anti-inflammatory action through selective suppression of transcriptional events of a gene group of iNos and Il6, and but not another group of Tnf.
Metabolome analysis revealed that ECEM contained anti-inflammatory compounds, including embelin, gingerol, diallyl sulfide, and 16-alpha-hydroxyestrone (Table 1).It is well-known that the LPS-elicited inflammatory response is initiated by binding of TLR4 with LPS on the cell surface, followed by activation of intracellular machinery, particularly the nuclear factor (NF)-κB signaling system [8].Embelin is a plant benzoquinone derivative with antitumor and anti-inflammation effects [16] and was reported to inhibit NF-κB activation [17].Gingerol, the main pungent component of ginger used in traditional Chinese medicine, is known to have antiinflammatory properties.Gingerol inhibited NF-κB-mediated iNos expression and enzyme activity in macrophage-like cells, RAW264.7,by decreasing LPS-induced IκB-alpha degradation, resulting in reduced NF-κB activity [9].Diallyl disulfide (DAS) is a major sulfur compound in garlic and has anti-inflammatory effect [30].DAS suppressed iNos expression and nitric oxide production in LPS-stimulated RAW264.7 cells [31].16-Alpha-hydroxyestrone has estrogen-like properties and binds or interacts with the estrogen receptor (ER) [24], which regulates transcription factors by inhibiting NF-κB activity on several levels [32].ER was known to sup-pressIl6 expression through interaction with NF-κB [33].In a previous report of 17β-estradiol (E(2)-like Tan IIA), its inhibitory effect on expression of iNos and Il6 was observed in LPS-induced RAW264.7 cells in an ER-dependent manner [34].The estrogenic flavonoid, baicalein, also exerted an anti-inflammatory effect by inhibiting iNos and Cox2 mRNA expression in LPS-stimulated RAW264.7 cells through an ER-dependent pathway and through regulation of NF-κB activation [10].Thus, ECEM appears to regulate the inflammatory response by downregulating pro-inflammatory genes, including iNos, Il6, and Cox2.
Tnf is also a pro-inflammatory gene upregulated by activation of NF-κB signaling with LPS and other stimuli [35] [36].Interestingly, ECEM exhibited no inhibitory effect on upregulation of Tnf expression in LPS-stimulated RAW cells, whereas ECEM potently suppressed the upregulated expression of iNos, Il6, and Cox2.There have been a few reports regarding differential regulation of Tnf and Il6 expression: mRNA expression of Tnf and Il6 were regulated differently in LPS-stimulated macrophages under hyperthermia conditions [37]; The DASsuppressed nitric oxide production was independent of TNF in LPS-stimulated RAW264.7 cell [22]; Methanol extracts from Euphorbia cooperi significantly reduced the production of nitric oxide and IL-6, but not TNF, in LPS-stimulated RAW264.7 macrophages and reporter assays demonstrated reduced activator protein-1 activity, while NF-κB activity was not reduced [38].LPS-induced genes were divided into 2 groups, an activating transcription factor 3-independent group including Tnf and Il1 and an activating transcription factor 3-dependent group including iNos and Il6, by regulation with heat shock transcription factor 1 under high-temperature stress conditions [39].These data suggest that transcriptional events of pro-inflammatory cytokines are complicated and regulated by association with not only NF-κB but also other factor(s).Moreover, the Tnf and iNos groups are regulated by different manner in LPS-mediated upregulated expression of these pro-inflammatory cytokine genes.Thus, ECEM affected inhibition of LPS-upregulated gene expression of one group, including iNos, Il6, and Cox2, but did not affect expression of another group containing Tnf, Ccl7, and Dusp 2, although the detailed mechanism of differential regulation of ECEM remains unclear.ECEM appears to be a good reagent for investigating differential regulation mechanisms on gene expression of iNos and Tnf groups in LPS-stimulated cells.
Another characteristic function of ECEM was observed in upregulated gene expression of Cd1d1 and Abcb9, which participate in antigen presentation.Cd1d1 is a member of gene family specific to rodents, mice, and rats, and a homologue to human CD1D.CD1D is a non-classical major histocompatibility complex molecule and presents lipid antigens to natural killer T (NKT) cells.NKT cells can produce both Th1 and Th2 cytokines and have immunostimulatory action against infectious diseases, as well as against cancer [40].In the current study, little upregulation of Cd1d1 expression was observed in LPS-stimulated RAW cells.This likely caused LPS to indirectly upregulate CD1 expression through glycolipid autoantigen synthesis [41].An insufficient amount of LPS, at a final concentration of 10 ng/mL, was used in our cell culture experiments, as 1 mg/mL LPS was reported to be optimal for upregulating Cd1d1 expression in LPS-treated RAW264.7 cells [42].These results demonstrated that upregulation of Cd1d1 expression is a characteristic function of lipid components but not contaminating LPS in ECEM.Another effect elicited by ECEM on the immune response was upregulation of Abcb9 expression.ABCB9 is also known as transporter associated with antigen processing-like, and translocates peptides from the cytosol into the lumen of lysosomes, indicating its involvement in antigen presentation in macrophages [43].Since ABC transporters, including the ABCB subfamily, are known to function in lipid translocation [44], Abcb9 likely has a synergic effect on Cd1d1-mediated lipid antigen presentation in macrophages.Thus, ECEM has unique immunomodulatory action to stimulate lipid antigen presentation by upregulating expression of both Cd1d1 and Abcb9.

Conclusion
In conclusion, we identified effective components in and biological functions of ECEM, a secondary metabolite produced by symbiotic culture with lactic bacteria and yeasts followed by photosynthetic bacteria, using metabolomic and functional genomic analyses.ECEM revealed unique anti-inflammatory functions to suppress LPSupregulated gene expression of one group of pro-inflammatory genes (iNos, Il6, and Cox2) but not of another group of genes (Tnf, Ccl7, and Dusp2).ECEM also showed characteristic immunostimulatory effects, upregulating the expression of Cd1d1 associated with antigen presentation on lipids and resulting in activation of NKT cells.These effects make ECEM a suitable material for developing a functional drink and/or food for health control.

Table 1 .
Major ingredients found in the ECEM and their biological roles ingredients.

Table 2 .
Genes up regulated by LPS and alleviation by ECEM to their LPS-up regulated expression.Gene set categorized as "Inflammatory response and/or immunoresponse" by Gene Ontology.Typical 25 genes.
* Negative fold change meaning inhibitory fold of ECEM to LPS-mediated up regulation.Genes highlighted in bold type were mentioned in discussion.

Table 3 .
Genes up regulated by LPS and no alleviation by ECEM to LPS-up regulated their expression.Gene set categorized as "Inflammation/Immune response" by Gene Ontology.Typical 6 genes.

Table 4 .
Genes up regulated specifically by ECEM but not LPS.Gene set categorized as "Antigen presentation" by Gene Ontology.Typical 2 genes.

Table 5 .
Genes downregulated specifically by ECEM but not LPS.Gene set categorized as "Intrinsic apoptosis" by Gene Ontology.Typical 5 genes.