MOLECULAR CELLULAR, AND DEVELOPMENTAL BIOLOGY :. High-throuhpüt expression analysis of intestinal intraepithelial lymphocytes after oral feeding of carvacrol, cinnamaldehyde, or Capsicum oleoresin

D. K. Kim,* H. S. Li1lehoj, 1 S. H. Lee,* S. I. Jang.* and D. Bravof *Animal Parasitic Diseases Laboratory. Animal and Natural Resources Institute, Beltsville Agricultural Research Center, USDA, Agricultural Research Service ; Beltsville, MD 20705; and tPancosma SA, CH-1218 Geneva, Switzerland

ABSTRACT Among dietary phytonutrients, carvac- seen in the Capsicum-fed broilers with 98 upregulated rol, cinnarnaldehyde, and Capsicum oleoresin are well and 156 downregulated compared with untreated known for their antiinflarnmatory and antibiotic effects controls. Results from the microarray analysis were con- inhuman and veterinary medicine. To further define the firmed by quantitative real-time PCR with a subset of molecular and genetic mechanisms responsible for these selected genes. Among the genes that showed >2.0-fold properties,-. broiler chickens were fed a standard diet altered mRNA levels, most were associated with meta- supplemented with eithei- of the 3 phytochemicals and bolic pathways. In particular, with the genes altered by intestinal intraepithelial lymphocytes were examined Capsicum oleoresin, the highest scored molecular net- for changes in gene expression by microarray analysis. work included genes associated with lipid metabolism, When coinped with chickens fed a nonsupplernented small molecule biochemistry, and cancer. In conclusion, standard diet, carvacrol-fed chickens showed altered ex- this study provides a foundation to further investigate pressipn of 74 genes (26 upregulated, 48 downregulat- specific chicken genes that are expressed in response to ed) and cinnamaklehyde led to changes in the levels of a diet containing carvacrol, cinnarnaldehyde. or Capsi- mRNAs corresponding to 62 genes (31 upregulated, 31 cum oleoresin. downregulñted). Most changes in gene expression were Key words: chicken, microarray, gene expression, metabolism . 2010 Poultry Science 89:68-81 doi: lO.3332/ps.2009-OO275

INTRODUCTION nets in vivo is much lower than those used in vitro. Therefore,the question remains of the mode of action The use of antibiotics as growth promoters in feed of these extracts within the range of doses which are has ..bèn ciiticized for the possible effects of agricul- used for growth-promoting purposes. It is critical to ture-related antibiotics on .the emergence of antibiotic obtain this knowledge to find the best way to utilize resistance in human pathogens. Many countries are these additives. now producinggnsiificant amounts of poultry without The dietary effects of the mixture of 3 phytochemi- antibiotic growth promoters even if the national regu- cAls, carvacrol, cinnamaldeh yde. and Capsicum oleores- lation still authorizes theinuse. This evolution encour- in, as antibacterial and antifungal agents have been re- aged resetfrchers to find alternative growth-promoting ported (Jamroz et al., 2005). Carvacrol is a component feed ddities Plant'ètrhcts aie gaining interest as a of numerous aromatic plants, such as Origa.num vuig are, feed additive (Greathead, 2003; Benchaar et al., 2008). thyme, and wild bergamot (Dc Vincenzi et at., 2004). The-efficacy of such products has been demonstrated The antimicrobial functions of these herbs are associat- especially in poultry (Hernandez et al., 2004); however, ed with carvacrol (Burt et al., 2005) because carvacrol their mode of action is still speculative. Many people vapor has been shown to inhibit Salmonella growth in argue a bactericide effect of essential oils known since chickens (Burt et al., 2007). Cinnamaldehyde is a con- ancient times, but the optimum dose of these prod- stituent of cinnamon and widely applied as flavoring. It has been proven to have strong antibacterial activity against Escherichia coli, Pseudomonas aeniginosa. En- 02010 Poultry Science Association inc. terococcus faecalis, Staphylococcus aureus, Staphylococ- Received June 1, 2009. Accepted September 20 : 2009. cus epidermidis, metliicillin-resistant Staphylococcus au- 'Corresponding author: [email protected] reus, Klebsiella pneurnoniae, Salmonella sp., and Vibrio

68 GENE EXPRESSION IN INTESTINAL INTRAEPITHELIAL LYMPHOCYTES parahemolyticus (Chang et al.. 2001). The hot pepper signed to 4 groups (5 birds/group). Carvacrol, cinnam- (Capsicum spp.) is a vegetable of importance in human aldehyde, and Capsicum oleoresin were obtained from nutrition and has many beneficial effects on human Pancosma S.A. (Geneva, Switzerland), Capsicum oleo- healthc (Conforti et al., 2007; Oboh et al., 2007). Capsi- resin (Pushp Brand Spices, Munirnji Foods and Spices um oleoresin, prepared by organic extraction of pepper Pvt. Ltd., Indore, India) was extracted from crushed fruits, contains antibacterial activity and is effective in Capsicum annuum fruits with volatile solvents lead- treating stomach illnesses (Spices Board, 2008). It con- ing to an oleoresin and finally processed to produce a tains the pungent principle capsaicm, which has effects powder. Carvacrol and cinnamaldehyde were made by on the resistance to Salmonella Enteritidis infection by synthesis. All diets were formulated to meet the nutri- altering p11 and histological changes (Tellez et al., 1993; ent requirements for chickens as recommended by the McElroy et al., 1994). NRC (1994). The proportions of each phytonutrient in Recently, application of high-throughput genomics feed were based on the optimal doses from our pretrial technology to nutritional science is providing novel in- experiments in which these doses showed optimum pro- formation concerning the molecular and genetic mecha- tective immune response against oral coccidiosis chal- nisms of dietary modulation of host immunit y, physiol- lenge (unpublished data). ogy, and metabolism. This emerging field of functional Chickens were fed for 7 d beginning from hatch with nutritional genomics, called nutrigenomics, has created a standard diet alone (control) or with diets supple- unprecedented opportunities for increasing our under- mented with carvacrol (5.0 mg/kg), cinnamaldehyde standing of how nutrients modulate gene and (3.0 mg/kg), or Capsicum oleoresin (2.0 mg/kg). The expression to influence cellular metabolism. Large-scale composition and the concentration of each phytonutri- gene expression profiles provide enormous information ent in feed were shown in Table 1. on the interactions between nutritional stimuli and host genes. The microarray technique is an ideal tool for RNA Extraction and Aminoallyl-Labeled gene expression profiling of a large number of genes RNA Preparation in a single assay using tissue-specific array,. platforms. Future studies on the biological function of genes using After euthanization of the birds, intestines were cut inicroarray data will advance our understanding and longitudinally and washed 3 times with ice-cold Hanks' knowledge of the genetic regulation of host physiologi- balanced salt solution containing 100 U/niL of penicil- cal response by nutrients. lin and 100 mg/mL of streptomycin (Sigma, St. Louis, The application of the microarray technique to broil- MO). The mucosal layer of the entire intestine was ers fed with individual plant extract or essential oil will carefully scraped using a surgical scalpel and TEL were lead to better understanding of their mode of action isolated by Percoll density gradient centrifugation as in vivo. The present study was conducted to examine described previously (Min et al., 2005). Total RNA was alterations in gene expression iii intestinal intraepithe- isolated from pooled TEL representing an equal number lial lymphocytes (JEL) of chickens-that were fed diets of cells (5.0 x TO' cells) per bird from each treatment containing either carvacrol or cinnamaldehyde, or an group using Trizol (Invitrogen. Carlsbad, CA) and pu- oleoresin of Capsicum oleoresin. rified using the R.Neasy Mini RNA Purification Kit (Qiagen, Valencia. CA) according to the instruction of MATERIALS AND METHODS the manufacturer. In preliminary experiments, we de- termined that equal cell numbers and equal amounts Experimental Birds and Diets of total RNA were obtained per unit volume of gut mucosa (D. K. Kim, unpublished data). Aminoallyl- All experiments were approved by the Beltsville Ag- labeled RNA from TEL was prepared using the Amino riculture Research Center Small Animal Care and Use Allyl Message Amp TI aRNA Amplification Kit (Am- Committee. One-day-old broilers (Ross/Ross, Longe- bion, Austin, TX) according to the instruction of the necker's Hatchery, Elizabethtown. PA) were housed manufacturer. Two 20-wg aliquots of each aminoallyl- in Petersime starter brooder units and randomly as- RNA sample were fluorescently labeled with AlexaFlu-

Table 1. Three dietary phytonutrients used in this study Concentration in feed (mg/ Nutrient Source Composition kg) Company Carvacrol Synthesized compound 99% of carvacrol 8.0 Nature Chemicals (Hamburg, Germany) Cinnama.ldehydc Synthesized compound >97% of cinnunmidehyde ALYS Technologies SA (B,issigny-pres- 3.0 Lausanne, Switzerland) Capsicum Extract from Capsicum 6% of capsaicin and 2.0 Pushp Brand Spices (Indore. India) oleoresin awl man dihydrocapsaicin

70 KIM ET AL. or 555 or AlexaFluor 647 (Invitrogen) and labeled RNA rio were column-purified using the .RNA Amplification Kit 0 0 (Anibion). The RNA concentrations and labeling effi- CM 0 0 ciencies were determined spectrophotometricafly. tO Cl 0) 00

C, MirOroarray Hybridization : /. .0 The avian.IEIJ array (AVIELA) consisted of 10,1.62 00 0 spots representing elements from 3 sources: 1) cDNA o -ci

from chickenJEL (Min et al:, 2005), 2) imrnune-relat- .0 0) Cl r 0 .,-, Cl 0 — ed cDNA from the lipopolysaccharide-activated HD1 I CM CM Cl CM CM CM CM chicken macrophage cell line (Min et al., 2003), and 3) Q direct iPCR. clones of selected chicken cytokines and L OH chernokines (Min et al. 2005). Each element was du- Hp 0 -tb Hc 2c- o- 0 plicated on the array slide. Six microarray hybridiza- cJ 00 - 0 tions were performed. According to a. reference design 0'. -co 0 0 withdye swap (Mcshaiie et al. 2003) 1 4 values were oO EH H < H oI_ 00 H 0 obtained for each treatment, 2 on one slide and 2 on the

dyes The scaiined:microarray images for each channel .4.-

were overlaid and fluorescent intensities were quantified Cd 2 o- CCC') CO 6') C' - F-0 t-t- 0' 60 0 - COO 00 Cl 0 CM using ScanArray Express version 3.0 software (Perkin- 0CM 0 CM 0 Elmer). Spots were detected , using an adaptive circle 5 00 algorithm in the ScanArray program and all spots were visually confirmed. I C, C, Microarray Data Analysis 4- 0 0 C, =. no " ri] C -e C) a, CeneSpring OX ,7.3 software (Silicon Genetics, Red- Z0 wood, CA) was used to qualify and normalize image t 0. nC analysis data and to perform the fold-change analyses. Median signal intensities were qualified by subtract- 00 . ing - the median - local background. and normalized by a, -C 22% block locally weighted regression and smoothing scatter F- P 5C. 0 o -c a plots methods. Flag information was applied to filter o bad spots with genes missing more than 50% of their 0 0 values because of a bad signal-to-noise ratio being re- to moved. To generate signal ratios, signal channel values 0 CM (treatment group) were divided by control channel val- C, -t -t C 8) 0 ues (control group). The significantly differentially cx- CM I- 0 Cx) a 00 0 0 0 00 Ht 0 0 0

GENE EXPRESSION IN INTESTINAL INTRAEPITHELIAL LYMPHOCYTES 71 Signal transduction Protein metabolism and modification Immunity and defense Intracellular protein traffic Other metabolism Nucleoside, nucleotide, and nucleic acid metabolism Phosphate metabolism Transport Developmental processes Miscellaneous Lipid, fatty acid, and steroid metabolism Cell cycle Homeostasis Carbohydrate metabolism Oncogenesis Apoptosis Cell structure and motility Biological process unclassified

0.00% 5.00% 10.00% 15.00% 20.00% 25.00% Percent of gene hit against total # genes

Figure i. (GO) analysis of genes mapped for human exhibiting >2,0-fold up- and downregulated expression after 7 d of dietary supplementation with carvacro]. The GO terms were based on the PANTHEI1, databases (litti)://www.1)atitliercil).org).

genes were filtered using the volcano plot built Bloinformatics naiysis by comparing the treatment of each phytonutriertt with itself. This volcano plot method is a statistically rigor- All sequence data files were obtained from the com- ous approach to quantifying microarray expression data piled database of the National Center for Biotechnol- that allows the relative effects of multiple treatments to ogy Information (NCBI). The IEL eDNA elements that be compared and incorporates analytical methods that were used to create the TEL cDNA microarray were are common to quantitative genetics (Jin et al., 2001). mapped to the chicken genome reference assembly (ver- The modulated elements were defined by 2-fold differ- sion 2.1) and reference RNA and protein sequences (for- ences and a cutoff of P < 0.05 by parametric test. The matted database for BLAST, May 2006) using NCBT microarray information has been submitted online into BLAST (version 2.2.13). To analyze pathway informa- the Minimum information About a Microarray Experi- tion, chicken gene identification numbers (ID) ment (http://www.mged.org/Workgroups/MJA ME!). that were ctifferentially expressed >2.0-fold between the The accession number for this stud y is E-MEXP-2204. treatment and control groups were mapped to Homolo-

Thble 3. Gene expression profiles induced by phytonutrients' Dietary supplement Upregulated Downregulated Total

Carvacrol (5 mg/kg) 26 48 74 Cinnamaldehyde (3 mg/kg) :31 :t 62 capsicmn annuum oleoresin (2 mg/kg) 98 156 254 'Chicken intraepithelia] lymphocyte (1EL) transcripts altered >2.0-fold using the avian IEL array after dietary supplemnentatiorm and compared a'itli a nonsupplemenl;ed diet.

72 KIM ET AL. gene ID (locus link ID) for human genes because a large Networks of focus genes were algorithmically generated portion of these sequences have not been defined in based on their connectivity. chicken. The mapped human gene ID were used for the classification by the terms of Gene Ontology (GO) from Quantitative Real-Time PCR the PANTHER database (http://www.pantherdb.org). Annotation derived from orthologous human genes al- To confirm gene expression changes observed by mi- low's for cthsssecies comparisons between chicken and croarray analysis, quantitative real-time PCR (qRT- human array data (Smith et at, 2006). PCR) was performed as described (Hong et al., 2006). The networks of: genes that were differentially ex- Equivalent amounts of the same R.NA samples used pressed by dietary Capsicurn oleoresin were analyzed by for microarray hybridizations were reverse-transcribed the Ingenuity Pathways Analysis software (Ingenuity using the StrataScript First Strand Synthesis System Systems, Redwood City, CA). The data set containing (Stratagene. La Jolla, CA). Amplification and detec- gene identifiers mapped to Homologene ID and cor- tion were carried out with the Mx3000P system and responding expression values were uploaded into the Brilliant SYBR Green qRT-PCR master mix (Strata- application. Each identifier was mapped to its corre- gene). Standard curves were generated using 1og10-di- sponding gene object in the Ingenuity knowledge base. luted standard RNA and levels of individual transcripts A fold-change cutoff of 2.0 was set to identify genes were normalized to those of glyceraldehyde 3-phosphate whose expression was significantly differentially regu- dehydrogenase by the Q-gene program (Muller et al., lated. These genes, called focus genes, were overlaid 2002). For the calculation of fold changes between con- onto a global molecular network developed from in- trol and treatment groups, the normalized cycle thresh- formation contained in the Ingenuity knowledge base. old values of the target gene to glyceraldehyde 3-phos-

Nucleoside, nucleotide, and nucleic acid metabolism

Protein metabolism and modification

Intracellular protein traffic

Carbohydrate metabolism

Transport

Phosphate metabolism.

Sensory perception

Cell cycle

Lipid, fatty acid, and steroid metabolism

Other metabolism (it 'u. 'I Signal transduction

Protein targeting and localization - Biological process unclassified

0.00% 5.00% 10.00% 15.00% 20.00% 25,00% 30.00% Percent of gene hit against total ft genes

Figure 2. Gene Ontoidgy (GO) analysis of genes mapped for human exhibiting >2.0-fold up- and downregulated expression after 7 d of dietary supplementation with cinnamaldehyde. The GO terms were based on the PANTHER databases (htt,p://wwxpantherdb.org).

GENE EXPRESSION IN INTESTINAL INTRAEPITHELIAL LYMPHOCYTES 73 Protein metabolism and modification Nucleoside, nucleotide, and nucleic acid metabolism Cell cycle Immunity and defense Signal transduction Cell proliferation and differentiation Lipid, fatty acid, and steroid metabolism Carbohydrate metabolism Developmental processes Intracellular protein traffic Transport Cell structure and motility Cell adhesion Apoptosis Miscellaneous Protein targeting and localization Amino acid metabolism Homeostasis Other metabolism Electron transport Phosphate metabolism Oncogenesis Neuronal activities Biological process unclassified

0.00% 5.00% 10.00% 15.00% 20.00% 25.00% Percent of gene hit against total # genes

Figure 3. Gene Ontology (GO) analysis of genes mapped for human exhibiting >2.0-fold up- and downregulated expression after 7 d of dietary supplementation with Capsicum oleoresin. ThA-GO terms were based on the PANTHER databases (http://wwupantherdb.org).

Table 4. Differentially expressed genes related to metabolism after dietary supplementation with earvacro[ compared with nontreated controls Human Gene Ontology category Gene name' Gene symbol Score gene ID' Carbohydrate metabolism Protein plmosphatase 1, regulatory (inhibitor) subunit 313 PPF1R3B 8.00E-106 79660 Lipid, fatty acid, and steroid Phospholipase A2, group HE PLA2G2E 0.00E+00 30814 metabolism Nucleoside, nueleotide, and nucleic acid Transcription factor 2. hepatic: LF-133: variant lmepatic nuclear TCF2 0.00E-i-00 6928 metabolism factor SET domain containing lB SETD1I3 0.00E+00 23067 Phosphate metabolism Solute carrier family 34 (sodium phosphate). member 2 SLC34A2 0.00E+00 10568 Serine-threonine kinase 25 (STE20 homolog. yeast) STK25 0.00E+00 10494 Protein metabolism and modification Selenoprotein X, 1 SEPX]. 2.00E-32 51734 Dual specificity phosphatase 1 DUSP1 0,00E+00 1843 Protease, serine. 2 (trypsin 2) PRSS2 0.00E-i-00 5645 Serine-thrennine kinase 25 (STE20 homolog, yeast) 511<25 0.00E•+00 10494 Other metabolism Coenzyme Q5 homolog, methyltransferase (Saccbarom.yces COQ5 0.00E+00 84274 ccrevisiae) Ornithine decarboxvlase antizyme 1 OAZI 0.00E+00 4946 Serine-threonine kinase 25 (STE20 homolog, yeast) STK25 0.00E+00 10494 'Gene names for human mapped by chicken Entrex gene identification nun,bens. = identification number. KIM ET AL. phate dehydrogenase were calibrated to the relevant of nucleoside, nucleotide, and nucleic acid metabolism, control line values. Oligonucleotide primers for qRTT 24 genes were modulated (2, 5, or 17, respectively); PCR are listed in Table 2. Each analysis was performed and in the category of immunity and defense-related in triplicate. genes: 13 genes were affected (ca±vacrol, 4; Capsicurn oleoresin, 9; Table 7). RESULTS Pathway Analysis of the Network Genes Gene Expression Profiles Induced by Phytönutrients Because the greatest number of alterations in TEL transcript levels was seen after the Capsicum oleoresin- Compared with untreated controls and using a cut- supplemented diet, further pathway analysis was per- off of >2.0-fold differential expression, the levels of 74 formed using the Ingenuity knowledge base to identify IEL mRNA were altered in carvacrol-fed chickens (26 the biological functions that were most significant to the upregulated, 48 downregulated), whereas cinnamalde- genes in the network. For this analysis, the genes were hyde-fed birds exhibited changes in 62 transcripts (31 matched with human gene counterparts and the human u regulated, 31 downregulated). The birds given the gene ID were used as the data set. In this analysis, all Capsicum oleoresin-supplemented diet displayed altered relationships between genes in the network are graphi- expression of 254 genes (98 upregulated, 156 downreg- cally represented as lines and nodes that are displayed ulated; Table 3). The genes changed by each dietary using various shapes representing the functional class supplementation of carvacrol, cinnamaldehyde, and of the gene product. These relationships are supported Capsicum oleoresin are listed in Supplemental Tables by at least one literature reference or from canonical 1 : 2, and 3, respectively (http://ps.fass.org/content information stored in the Ingenuity knowledge base. A 1'0189/issue1/). total of 10 biologically relevant networks were deter- mined in this study. The most significant network was GO Annotation and Bioinformatics Analysis composed of 35 focus genes [a,cyl-coenzyme A oxidase 1, palmitoyl (ACOX1); adipose differentiation-related The altered transcripts corresponding to human gene protein (ADFP); adiponectin (ADIPOQ); adiponec- ID were analyzed by the CO Index using PANTHER tin receptor 2 (ADTPOR2); cychn Dl (CCND1); CD36 databases (Mi et al,. 2005; Figures 1, 2, arid 3). Of the molecule (CD36); CD74 molecule (CD74); CDC5 transcripts corresponding to genes classified in the bio- cell division cycle 5-like (Schizosaccharomyces pombe) logical process branch, 76 were related to metabolism (CDC5L); collagen; cystatin C (amyloid angiopathy (carvacrol, 13; cinnamaldehyde, 1.2; Capsicum oleoresin, and cerebral hemorrhage) (CST3); cathepsin S (CTSS); 51; Tables 4, 5, and 6). In the specific category of protein eukaryotic translation elongation factor 2 (EEF2); ex- metabolism and modification. 24 genes were modulated tracellular signal-regulated kinase (ERK); fatty acid (carvacrol, 4; cinnamaldehyde, 3; Capsicum oleoresin, synthase (FASN); hexokinase 1. (HK1); insulin; kinesin 17). In the category of signal transduction, 15 genes family member SB (KIFSB); low-density lipoprotein were altered (5, 1, and 9, respectively); in the category (LDL); lectin, galactoside-binding, soluble, 3 (gaiectin

Table 5. Differentially expressed genes related to metabolism following dietary supplementation with cimmamaldehyde compared with nontreated controls Gene Human Gene Ontology category Gene name' symbol Score gene ID' Carbohydrate metabolism UDP glycosyltransferase S (UDP-galactose ceramide IJGTS O.Ol]E+OO 7368 galactosyltrajisferose) Oxoglutarate (u-ketoglutarate) dehydrogenase (lipoanide) OGDH O.00E±OO 4967 Lipid. fatty acid, and steroid UDI' glycosyltransferase 8 (UDP-galactose ceramide UCT8 0.00E+00 7368 metabolism galnctosyltransfensse) Nucleoside, nucleotide, and nucleic acid DEAD (Asp-GIn-Ala-Asp) box polypeptide 1 DDX1 0.00E+00 1653 metabolism Phosphodiesterase CC, cCMP-spccific, cone, a pilate Pl)EUC 0.00E+00 5146 Chromobox homolog 3 (HP1 gamma homolog. Drasophilo) CBX3 7.oOE-49 11335 CTD (carhoxv-terminal domain. RNA polvmernse II, polypeptide A) CTDP1 0.00E4-110 9150 phosphatase, subunit 1 SMYD family member 5 SMYDS O.00E+00 10322 Phosphate metabolism Serine-tbreonine kinase 4 STK4 O.00E+00 6789 Protein metabolism and modification Nucleotide binding protein 2 (MinD lLo,llolog. Eseherichia coN) NU]3P2 O.00E+[llJ 10101 Protease, serine. 2 (trypsin 2) PRSS2 0.00E+00 5645 Serine-threonine kinase 4 STK4 0.00E+00 6789 Other metabolism Serine-threonine kinase 4 STK4 0.00E+00 6789 'Gene mart for human mapped by chicken Entrez gene identification numbers, = identification number.

GENE EXPRESSION IN INTESTINAL INTRAEPITHELIAL LYMPHOCYTES 75

3) (LGALS3); v-maf musculoaponeurotic hbrosarcoma protein (tapasin) (TAPBP) threonyl-tRNA synthetase oncogene homolog B (avian) (MAFB); MHC class I; (TARS); transforming growth factor (TGF-0); ty- MRC class 11: nuclear receptor corepressor (N-cor); rosine 3-monoox ygenase/tryptophan 5-nionooxygentise N-myc downstream regulated gene 1 (NDRG1): oxo- activation protein, epsilon polypeptide (YWHAE); and gluta.rate (cv.-ketoglutarate) dehydrogenase (lipoa.mide) zinc finger. MYNID domain containing 11 (ZMYND11)] (OCDFT); poly(A) polymerase a (PAPOLA); platelet- and a score of 56 (Figure 4). This first network of genes derived growth factor 0 polypeptide [simian sarcoma was associated with lipid metabolism (P-value: 2.45 viral (v-sis) oncogene homolog] (PDCF BB); protease. x io- to 2.47 x 102), small molecule biochemistry serine, 2 (trypsin 2) (PRSS2); prostaglandin E receptor (P-value: 2.45 x 10 9 to 2.47 x 10-2)7 and cancer (P- 4 (PTGER4); retinoid X receptor (Rn); TAP binding value: 3.50 x 10 5 to 2.89 x 10_2). The second network.

Table 6. Differentially expressed genes related to metabolism after dietar y supplementation with Capsicum oleoresin conipared with nontreated controls

Human gene Gene Ontology category Gene 'lame' Gene symbol Score ID' Amino acid metabolism Splmingosine-1-phosphate lyase 1 SC FL 1 0.00E+OO 8879 Fatty acid svntha.se FASN 0.00E+OO 2194 Carbohydrate metabolism Ghitobiase, di-NN-acetyl- Cr135 O.00E+OO 1486 Protein phosphatase 1, regulatory (inhibitor) subunit 313 PPP1FI3B SOUL-lOG 79660 Hexokinase I HKI 0.00E+0O 3098 Oxoglntarate (a-ketoglutarate) delivdrogenase (lipoamnide) OG DH O.00E+liO 4967 Adiponectin, G1Q and collagen domain containing ADIPOQ 1)005+00 9370 Phosphoglycerate mnutase family member 4 PtIA?v14 0.00E+o0 441531 Lipid 7 fatty acid, and steroid Adipose differentiation-related protein ADFP 01105+00 123 metabolism Fatty acid synthase FIN N 0.00 5±00 2194 Gytochromne P450, funiily 2. snhfannlv J. polypeptide 2 CYP2J2 1)005±00 1573 Acyl-coenzyine A oxidase I., palnñtoyl AC OX 1 0.005-1-00 51 Adiponectin receptor..2.. ADIPOR2 0.00E+00 79602 Adiponectin. C1Q and collagen domain containing ADIPOQ 0.005±00 9370 Nucleoside, nucleotide. and CGR.4-NOT transcription complex, subunit I CNOT1 0.UOE+00 23019 nucleic acid metabolism GDG5 cell division cycle 5-like (Schizoso echo raropees ponthe) CDGSL (1.005-4- 00 988 Splicing factor 3b. subunit 4, 49 kDa SF3134 0.00E+Ol) 10262 Splicing factor prolinc/gluta.mine-ricb (polypyrinudine tract binding SFPQ 0.1105+00 6421 protein associated) ?vIYST histone acetyltransferase 2 MYST2 0,00E+00 11143 Transcription elongation regulator 1 TGERCI 0.00E+00 10915 Exosome component. i EXOSCI 2.005-87 51013 Zinc linger. MYND domain containing 11 ZMYND11 0.005+00 10771 v-maf musculoaponeurotic librosarcoma oncogene homolog B (avian) S'IAFB 0. OOE-± 00 9935 Heterogeneous nuclear rihonucleoprotein A3 Ii NRPA3 0.005+00 220988 DEAD (As1sClu-Ala-Asp) box polypeptide 10 DDX1O 0.005+011 1662 Threommyl-tR.NA syntlmetase TARS 0.005-1-00 6897 Translin ]SIN 0.005-4-00 7247 FUS interacting protein (serine-arginine-rich) 1 FUSIP1 5.1105-93 10772 Cleavage stimulation factor, 3' pre-RNA. subunit 2, 64 kDa. Tan variant GSTF2T 0.005+00 23283 DEAD (Asp-GIn-Ala-Asp) box polypeptide 18 DDXI8 0.005+00 8886 Poly(A) polynierasc 0 PAPOLA 0.005+00 10914 phosphate metabolism Serine-threommimme kinase 25 (STE20 honmoiog, yeast) STK2S 0.005±00 10494 Protein metabolism and Unc-51-like kinase 1 (CoenorhabdiUs elegans) U 111< 1 0.005+00 8408 modification Eukaryotic translation initiation factor 3 1 subunit 6 interacting protein EIF3S6IP 0.005+00 51386 ATG4 autophagy related 4 homolog B (Saccharvroyces cerevisiae) ATC4B 0.00E+00 23192 MYST histono acetyltransferase 2 m\IYST2 0.005+1)0 1114:3 Uhiqoitin-conjugating enz yme E21 (0BC9 homulog. yeast.) UBE21 3.005-li 5 7329 Asparaginyl-tENA synthetasc NAIlS 0.005+00 4677 Protease. serine, 2 (trypsin 2) P11552 0.00E+00 5645 Eakaryotic translation elongation factor 2 EEF2 0.005+00 1938 Gysta.tin C (amyloid angiopathy and cerebra! hemorrhage) CST3 7.005-83 1471 Hibosornal protein U) RP.L9 0.005+00 6133 Procollagen-lysine I, 2-oxoglnt.a.ratc 5-dioxygenase 1 PLODI 0.005+00 5351 Cat hepsin S CTSS 0.005+00 1520 Threonyl-tRNA synthetase TARS 0.005+00 6897 Calcium binding protein P22 CHI, 7.005-120 11261 Stroma) cell-derived factor 2-like I SD F2L I 7.005-96 23753 Serino-tlmrennine kinase 25 (STE20 homolog. yeast) 5TK25 0.005+00 10494 Chymotrypsinogen 132 GT11132 0.005+00 440387 Other metabolism Stromal cell-derived factor 2-like 1 SDF2LI 7.005-96 23753 Serine-threonine kinase 25 (STE.20 Imomnulog, yeast) STK25 0.005+00 10494 'Gene names for human mapped by chicken Entrez gene identification numbers. 51D = identification mu mniher.

76 KIM ET AL. regulated by 17 focus genes with .a score of 31, was re- >2.0-fold altered expression in the normalized AVIELA lated with cellular assembly and organization (P-value: data (P < 0.05). Of these, 2 (CD74 and CDC5L) were 3.46 x io to 2.94 x 102). gastrointestinal disease associated with the first network and 3 (UBE2I, CD247, (P-value: 3,50 x 1070 to 2.36 x 10_2), and genetic dis- and FADD) were included in the second network of order (P-value: 2.56 x 10" to 2.94 x 10_2) (Figure 5). pathway analysis. As shown in Figure 6, the transcrip- The second network of genes included actin; V-akt mu- tional changes in these genes as assessed by qRT-PCR rine thyinoma viral ohcbiei h6molog (Akt); activator showed similar patterns when compared with the origi protein '1 (Api); caspase; CD247 molecule (CD247); nal microarray data. calciumbinding protein P22 (CHP); cAMP response element binding .(Creb); colony stimulating factor 3 DISCUSSION receptor (granulocyte) (CSF311); cytochrome P450, family 2, subfamily J, polypeptide 2 (CYP2J2); Fits The AVIELA microarray used in this study was origi- (TNFRSF6)-associated via death domain (FADD); nally described for its ability to characterize gene ex- follicle-stimulating hormone (FSH): historic 113; his- pression in TEL of chickens infected with the intestinal tone 114; 'hematological and neurological expressed I apicoinplexan protozoa Eimeria, the etiologic agent of (HN1); interferon-o; c-Jun N-terminal kinase (JNK); avian coccidiosis (Allin et al. 2003, 2005; Kim et al. mitogen-activated protein kinase (MAPK); MYST 2008). Because the mucosal layer of the intestine not histone acëtyltransfera.se 2 (MYST2); nuclear factor only plays an important role in the immune defense of activated T-cells (NFAT); nuclear factor r,-light- against ingested pathogens but also comes in direct chain-enhancer of activated B cells (NF-KB); phospho- contact with foods and nutrients, we used the AVIELA inositide 3-kinase (P13K); prostaglandin-endoperoxide as a surrogate tool for gene expression profiling dur- synthase 2 (PtGS2); Rita protein (Ras); RHOC = ing digestive and absorptive processes in the chicken Ras homology growth-related; RNA polymerase Ii; gut. The utility of this approach was verified by the splicing factor 3b. subunit 4 (SF3134); splicing factor fact that the results from, the microarray analysis were proline/glutamine-rich (polypyrimidine tract binding confirmed by qRT-PCRf, The small differences in the protein associated) (SFPQ); signal recognition particle magnitude of the changes observed by inicroarray and 72 kDa (5RP72); serine-threonine kinase 25 (STK25); qRT-PCR might be due to differences of the normaliza- spleen tyrosine kiiase/zetachain-associated protein ki- tion methods used by the 2 approaches or the different nase; transcription elongation regulator I. (TCERG1); fluorescent dyes used, or both (Lee et al. 2002). ubiquitin-conjugating enzyme E21 (UBE2I); ubiquitin; This trial demonstrated that 5 mg/kg of carvacrol al- WAS/WASL interacting protein family, member 1 tered expression of 74 genes (26 upregulated, 48 down- (1VIPF1); and zvxin (ZYX). regulated), 3 mg/kg of cinnamaldehyde led to changes in the expression of92 genes (31 upregulated, 31 down- qRT-PCR regulated), and 2 mg/kg of Capsicum oleoresin led to alterations in 254 genes expression (98 upregulated, To confirm the microarray analysis, we selected 7 156 downregulated) compared with untreated controls. genes and followed the kinetics of their corresponding These results suggest that among these 3 phytonutri- transcript levels after dietary supplementation with ents, Capsicum oleoresin is the strongest modulator of Capsicuni oleoresin. All of the selected genes showed transcriptional control in chicken IEL.

Table 7. Differentially expressed genes related to immunity and defense after dietary supplementation with carvacrol or Capsicum oleoresin compared with no,,treated controls Human gene Nutrient Gene name' . . Gene symbol Score ID' Carvacrol -Selenoprotein X, 1 SEPX1 2.00E-32 51734 Cheinokine (C motif) ligand 2 XGL2 6.00E-65 6546 Leucine-rich repeat containing 59 LRRG5Y O.00E-I-OO 55379 CO5 niolecule CD5 0.00E+00 921 Capsicum oleoresin Lectin, galactoside-binding. soluble. 3 (galectin 3) LGALS3 7,00E-59 3938 9 open reading frame 58 G9or158 2.00E-1Q8 83543 TAP binding protein (tapasi,,) TAPI3P 0.00E+00 6892 GD247 molecule GD247 6.00E-121 919 Colony stimulating factor 3 receptor (granulocyte) GSF3R 0.0013+00 1441 GD74 molecule, MEG. class IT invariant chain GD74 0.00E+O0 972 Cathepsth S CTSS 0.00 E+00 1520 Translin TSN 0.0013+00 7247 Prostaglandin-endoperoxide synthase 2 (prostaglandin C/H synthase and PTGS2 0.0013-1-00 5743 - cyclooxygenase) 'Gene names for human mapped by chicken Entrez gene identification numbers. 'ID = identification number.

GENE EXPRESSION IN L\l'i: STINAL INTRAEP1I11F .l\L TX\IPI IO('YTES I1

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,

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Figure 4. First network of genes exhibiting >2.0-fold up- and downregulated expression levels after 7 d of dietary supplementation with Capsicum oleoresin. The intensity of each gene indicates the expression level of the genes. Up- and downregulated genes are illustrated with red and green colors, respectively. OGDH = oxoglutarate (a-ketoglutarate) dehydrogenase (lipoanude); TAPBP = TAP binding protein (tapasin): PTCER4 = prostaglandin E receptor : LGALS3 = lectin, galactoside-binding. soluble. 3 (galectin 3); MAFB = v-maf musculoaponeurotic fibrosarcoma olicogene hornolog B (avian); ERK = extracellular signal-regulated kinase; TGF-3 = transforming growth factor 3: YWHAE = tyrosine 3-nionooxygenase/tryptophan 5-monooxygenase activation protein, epsilon polypeptide: PDGF BB = platelet-derived growth factor 3 polypeptide [simian sarcoma viral (v-sis) oncogene homolog]; HK1 = hexokinase 1: PAYOLA = poly(A) polymerase n: LDL = low-density Ii- poprotein; CD36 = CD36 molecule: CCND1 = c yclin Di; PRSS2 = protease, serirle. 2 (trypsin 2); Rxr = retinoid X receptor: ADFP = adipose differentiation-related protein; ADIPOQ = adiponectin: ADIPOR2 = adiponectin receptor 2: FASN = fatty acid synthase; EEF2 = eukaryotic translation elongation factor 2; ACOX1 = acyl-coenzymeA oxidase 1. palinitoyl; N-cor = nuclear receptor corepressor: NDRG1 = N-myc down- stream regulated gene 1: TARS = threonyl-tRNA synthetase: CDC5L = CDC5 cell division cycle 5-like (Schizosaccharomyces pombe); KIF.5B = kinesin family member SB: ZMYND11 = zinc finger. MYND domain containing 11: CTSS = cathepsin S; CD74 = CD74 molecule; CST3 = cystatin C (amvloid angiopathy and cerebral hemorrhage).

Capsicum oleoresin is obtained from pepper plants as tiinflarnmatory activities, and inhibits the progression of a mixture containing capsaicinoids, including the active early or late stage tumors (Beltran et al., 2007). Finally. ingredient capsaicin, and other compounds. Further- Capsicum oleoresiri increased pancreatic and intestinal more, capsaicin has been reported to influence various enzyme activity (Platel and Srinivasan, 2000) and bile metabolic processes (Miller et al., 1983). Capsaicin and flow and bile acid secretion (Abdel Salam at al., 2005). capsaicinoids have been reported for their effects on the Within the 254 genes altered by feeding Capsicum oleo- gastrointestinal tract and animal nutrition (Nopanitaya. resin, the most significant biologically relevant network 1974). Sainbaiah and Satavanarayana (1989) have pos- activated by Capsicum oleoresin intake was composed tulated that capsaicinoids counteract the accumulation of 35 focus genes that encode for gene products related of fat in the liver by the reduction of hepatic lipogenesis to lipid metabolism, small molecule biochemistry, and or increased oxidation of lipids, or both. Capsaicin also cancer. In chickens. capsaicin. cinnamaldehyde, and induces neurogenic inflammation, has analgesic and an- carvacrol induced lipase activity in the pancreas and I'I\l LI \L.

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Figure 5. Second network of genes exhibiting >230-fold up- and dowuregnlated expression levels after 7 ci of (hetarv snppleineritatioii with Cap- srciLia oleoresiri. The intensit of each gene ind ic at e,, the expression level of the genes. Up- and downregulaterl genes are illustrated with red and icui colop, ii sp ( to rl\ Zs N = 7\ xin RHO( = is howolog grow th-related: Ct P212 = C\ toc litiOn( P450. famil y 2 suht rinfl I poly ) cp tide 2: C'HP = calennn binding protein P22: HNI = hematological and neurological expressed 1: FADI) = Faa (TNFRSF6)-a.ssociated via death domain: SRP72 = signal recognit ion particle 72 kDa: .JN1K = c-Jun N-terminal kinase: Akt = V-akt niuriure thyniotna viral oncogene hornolog: MAPK = rnitogen-activated protein kniase: SFPQ = splicing factor proline/glutrunine-rich (polvpvrimidine tract binding protei n associated); NF- r.13 = nnclear factor uc-light-chain-enhruic'er of activated B cells: NFAT = nuclear factor of activated I-cells: CSF3R = colony stimulating factor 3 receptor (granulocyte): P13K = phosphoinositide 3-kinase: PTGS2 = prostaglandin-encioperoxide svnthiasu.' 2: Api = activator protein 1: Creb = cAMP response elenoent liotiling; TCERG1 = transcription elongation regulator 1: SF3B4 = splicing factor 31). suburut 4; \VIPFI = WAS/ WASL interacting protein faniilv. member 1: FSI-I = follicle-stimulating hormone: CD247 = CD217 molecule; STK25 = serine-tln-eonine kina.se 25: MYST2 = MYST historic acctvltransferase 2: UBE2I = nhiquitin-conjugating enzyme E21: Has = Ras protein: SYK/ZAP = SYK/ZAP = spleen tyrosine kinase/zeta-chain-associated protein kinase.

intestine wall (Jarnroz et al., 2005). Therefore, it was and is relatively more abundant compared with other suggested that they played a critical role in the lipid plasma hormones (Diez and Iglesias. 2003). Cyclin Dl metabolic mechanism. Among these gene products, (CCND1) has been shown to interact with the tumor CD36 is a cell surface receptor that binds to a variety of suppressor protein retinoblastonia (Rb: Kishimoto et components, including collagen (Tainloii et al.. 1989). al., 2008) and mutation of its corresponding gene may thrombospondin (Silverstein et al.. 1992), erythrocytes contribute to tumorigenesis (Barbash et al.. 2008). The parasitized with Plasmodium falciparum (Oquendo et second most significant network identified in this study al., 1989), oxidized low-densit y lipoprotein (Nicholson was composed of 17 focus genes that encode et al.. 1995), native lipoproteins (Calvo et al.. 1998), associated with cellular assenibly and organization, oxidized phospholipids (Podrez et al., 2002), and long- gastrointestinal disease, and genetic disorder. In this chain fatty acids (Baillic et al., 1996). Adiponectin network. PTGS2 has been reported to be expressed at (ADIPOQ) is a protein hormone that modulates sever- significantly higher levels iii malignant, gastrointestinal al metabolic processes, including glucose regulation and stronial tumors compared with benign tuniors (Miao ct fatty acid catabolism. In addition. ADIPOQ is exclu- al., 2008). The zeta polypcptide chain of CD247 plays a sively secreted froni adipose tissue into the vasculature key role in signal transduction after antigen recognition (;1\V IXllIHlO IN !ll:*II\.\I, lFk\l : 1)illll : lI\l . l\lllIo(Yll

2.5p

0

N 15 D Microarray 0 z n. i .RT-PCR 0.5

:si $ fiLl c? '< c? I CP Figure 6. Comparison between the expression levels of selected genes from microarray analysis and quantitative real-time PCB (1IT-P('R) alter (lietarv supplementation with Capsd-nrn oleoresin. CD7l = CD74 molecule: I'SN = translin: UBE2I = ulaquitin-conjugating enz yme E21: CDC. L = CDC5 cell division cycle 5-like; CDK5RAP2 = CDK5 regulatory summit associated proteill 2; FADD = Fas (TNFRSFG)-associated via death domain: CD247 = CD247 miolecule. by the T-cell receptor, and its clowriregulatiori might regulated. Hemoglobin. 3 (HBB) is an oxygen trans- be responsible for deficient cellular innnune responses porter (Them et al.. 1990) and a mutation in HB[3 (Eleftheriaclis et al., 2008). causes sickle cell anemia (Persons. 2003). Within the 74 genes that expressed significant al- Generally, the supplementation with carvacrol, ciii- teration after feeding 5 mg/kg of carvacrol, several imanialdehyde, and Capsicum had no influence on B\V genes related with the endocrine and metabolic system or feed efficiency, but these supplements improved ileal such as selenoprotein X, 1 (SEPX1) and protease, ser- and fecal digestibility in homeostatic status in chicken,,, me, 3 (PRSS3) were upregulated. Selenoprotein X, 1 (McElroy et al., 1994; Hernandez et al., 2004: .lamroz (SEPX1) belongs to the methionine sulfoxide red ict.ase et al., 2005). The detailed mechanisms are not known B family and has high specificity for reduction of the but may involve morphological modification of cells of R forms of free and protein-bound rnetlnonine sulfoxide the gastrointestinal nnucosa (.lamnroz et al.. 2006) and (Moskovitz et al.. 2002). Protease. serine. 3 (PRSS3) genetic regulation of metabolic network. Furthermore. encodes inesotrvpsin in humans and has the property challenge of pliytonutrient-fed birds with Eimei'ia re- of resistance to biological trypsin inhibitors, such as duced gut lesion, enhanced BW, and decreased fecal pancreatic trypsin inhibitor, so bean. lima bean. ovo- oocvst output. In addition, the local production of inucoid inhibitor, and n 1-antitrvpsin (Rinderkneclit et 1roiniffiuuuirit or cvtokines was significantly decreased. al., 1984). It might be from the biological function of In our results. several immune-related genes showed digestive degradation of trypsin inhibitors (Szinola et changes after treatment with pliytomitricnts reflecting al., 2003). Carvacrol has well-known antibacterial and their well-known medicinal effects against various bac- antifungal properties as well as antioxidative effects, teria or fungi in chicken infection studies (Tellez et al., with several potential applications for topical treat- 1993: Janiroz et al.. 2006; Burt et al.. 2007). Clearly, ment of niucosal and epithelial infections (Chanii et these phvtonutrients exert significant effects on host, al.. 2004a.1): Kristinsson et al., 2005: Burt et al.. 2005). immunity, metabolism, and physiology by altering the The antiinflaminatory properties of carvacrol have been expression of important genes associated with host dis- suggested to he due to inhibition of inducible PTGS2 ease resistance against pathogens. isoforins (Landa et al., 2009), a gene we found to he III transcriptional profiling and pathway downregulated after dietary supplementation with Cap- analysis revealed differential expression by 3 dietary and sicum oleoresin (Supplemental Table 3: littp: //ps.fass. phiytonutrients identified network of genes induced org/content/vol89/issuel/). by Capsicum oleoresin. These results provide new infor- Among the altered genes after feeding 3 mg/kg of mation concerning the molecular mechanism.-; involved cinnamaldehyde, hemoglobin, 3 (HBB) was highly up- in dietary modulation of host immunity, physiology.

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