Gene Expression Analysis of the IPEC-J2 Cell Line: A

Hindawi Publishing CorporationISRN GenomicsVolume 2013 Article ID 980651 7 pageshttpdxdoiorg1011552013980651

Research ArticleGene Expression Analysis of the IPEC-J2 Cell Line A SimpleModel for the Inflammation-Sensitive Preterm Intestine

Ann Cathrine F Stoslashy1 Peter M H Heegaard1 Per T Sangild2

Mette V Oslashstergaard2 and Kerstin Skovgaard1

1 Innate Immunology Group National Veterinary Institute Technical University of Denmark Bulowsvej 27DK-1870 Frederiksberg C Denmark

2Department of Nutrition Exercise and Sports University of Copenhagen Rolighedsvej 30DK-1958 Frederiksberg C Denmark

Correspondence should be addressed to Ann Cathrine F Stoslashy acfstvetdtudk

Received 31 January 2013 Accepted 20 February 2013

Academic Editors H Fuchs and P Wigley

Copyright copy 2013 Ann Cathrine F Stoslashy et al This is an open access article distributed under the Creative Commons AttributionLicense which permits unrestricted use distribution and reproduction in any medium provided the original work is properlycited

The IPEC-J2 cell line was studied as a simple model for investigating responses of the newborn intestinal epithelium to dietsEspecially the small intestine of immature newborns is sensitive to diet-induced inflammation We investigated gene expressionof epithelial- and immune response-related genes in IPEC-J2 cells stimulated for 2 h with milk formula (CELL-FORM) colostrum(CELL-COLOS) or growth medium (CELL-CONTR) and in distal small intestinal tissue samples from preterm pigs fed milkformula (PIG-FORM) or colostrum (PIG-COLOS) High throughput quantitative PCR analysis of 48 genes revealed the expressionof 22 genes in IPEC-J2 cells and 31 genes in intestinal samples Principal component analysis (PCA) discriminated the geneexpression profile of IPEC-J2 cells from that of intestinal samples The expression profile of intestinal tissue was separated by PCAinto 2 groups according to diet whereas no diet-dependent grouping was seen for IPEC-J2 cells Expression differences betweenPIG-FORM and PIG-COLOS were found for DEFB1 CXCL10 IL1RN and ALPI while IL8 was upregulated in CELL-FORM com-pared with CELL-CONTR These differences between IPEC-J2 cells and intestinal tissue from preterm pigs both used as modelsfor the newborn intestine underline that caution must be exercised prior to analysis and interpretation of diet-induced effects ongene expression

1 Introduction

The intestine is the site for nutrient digestion and absorptionbut also a major immunological defense barrier that recog-nizes and responds to external antigens In addition to the gutmucosal immune system intestinal epithelial cells (IECs) areinvolved in the initiation and coordination of the intestinalimmune response by the production of signaling moleculesincluding cytokines and chemokines [1]The IPEC-J2 cell lineoriginates from the jejunum of an unsuckled neonatal pig[2] and is morphologically and functionally similar to IECswith microvilli and tight junctions as well as expression andproduction of cytokines defensins toll-like receptors andmucins [2ndash4] It has previously been used to investigate host-pathogen interactions and immune responses with relevancefor human and swine intestinal diseases [2 3 5ndash8] and could

be a simple in vitromodel to investigate the immune responseof IECs in newborns

Necrotizing enterocolitis (NEC) is a serious gastrointesti-nal disease in preterm infants arising from the combinedeffect of prematurity abnormal bacterial colonization andenteral feeding In particularly feeding with milk formulapredisposes toNECwhereas humanmilk is protective [9 10]We set out to evaluate the IPEC-J2 cell line as an in vitromodelfor the preterm neonatal intestine and as a supporting modelfor the well-established preterm pig model of NEC whichis a valuable model for investigation of diet-induced effects[11ndash14] In the preterm pig model of NEC milk formula-fedpreterm pigs have more NEC lesions compared with pretermpigs fed porcine or bovine colostrum rich in growth- andimmuno-modulatory factors [11 15 16]

2 ISRN Genomics

Compared with animal models cell line studies are lesscost intensive associated with no ethical concerns and pro-vide a highly-controlled simple model to investigate isolatedfactors for example diet on the IECs response The IPEC-J2 cell line has already been shown to be a valuable model forthe investigation of host-pathogen interactions and could alsobe a promising model for in vitro studies of innate immunefunctions of neonatal IECs in response to dietary stimuliIn this study we evaluated the potential of the IPEC-J2 cellline as an in vitro model to study diet-induced effects on thepreterm neonatal intestine We analyzed and compared theexpression of epithelial- and immune response-related genesin the IPEC-J2 cell line and in preterm pig intestinal tissue

2 Materials and Methods

21 IPEC-J2 Cell Line Study IPEC-J2 cells [2] were main-tained inDulbeccorsquosmodified eaglemedium (DMEM)HamrsquosF-12 (1 1) supplemented with 10 fetal calf serum penicillin(100UmL) streptomycin (100 120583gmL) pyruvate (1mM)and L-glutamine (2mM) (growth medium Sigma-AldrichBroslashndbyDenmark) in a humidified atmosphere of 5CO

2at

37∘C Passage was performed approximately once a week andcells between passages 82ndash85 were used in the experimentPrior to the experiment the cell culture was found to be freeof mycoplasma

For the experiment cells were transferred to Corn-ing Transwell-COL collagen coated-membrane (04 120583m poresize Sigma-Aldrich) and grown in growth medium sup-plemented with epidermal growth factor (5mgmL SigmaAldrich) and insulin-transferrin-selenium (5 120583gmL of eachSigma-Aldrich) until stable transepithelial electric resistancevalues measured with an EVOMmdashEpithelial Voltohmme-ter (World Precision Instruments Berlin Germany) werereached after approximately 12 d The cells were stimulatedfor 2 h in three different diet solutions growthmedium alone(CELL-CONTR 119899 = 4) growth medium containing 1gamma-irradiated bovine colostrum (CELL-COLOS 119899 =4) or growth medium containing 1 milk formula (CELL-FORM 119899 = 4) These conditions were selected based onresults from preexperiments testing cell viability under dif-ferent concentration of colostrum and periods of time Thecolostrum and milk formula were identical to those used inthe pig study (see later) The lipid fraction and cellular debriswere removed by centrifugation at 3500 rpm for 20min at4∘C After stimulation the cells were gently washed twicewith Dulbeccorsquos phosphate buffered saline (D8537 Sigma-Aldrich) harvested and stored at minus80∘C

22 Preterm Pig Study Nineteen preterm pigs were deliveredfrom four sows by Caesarean section (Large White times DanishLandracetimesDuroc Askelygaard Farm Roskilde Denmark) at105ndash107 d of gestation (90ndash92 gestation) Procedures forCaesarean section and nursing of the preterm pigs followed astandard protocol previously described in details [11 12] Thepigs were given total parenteral nutrition through a vascularcatheter (4mLkgh advancing to 6mLkgh) for 2 d basedon Nutriflex Lipid Plus (Braun Melsungen Germany) and

adjusted in nutrient composition to meet the requirementsof pigs as described by [11] Hereafter pigs were randomizedaccording to birth weight into two enteral nutrition groupsgamma-irradiated (1 times 10 kGy Sterigenics Espergaeligrde Den-mark) bovine colostrum (PIG-COLOS 119899 = 6 kindly donatedby Biofiber-Damino AS Gesten Denmark) or milk formula(PIG-FORM 119899 = 13 80 g Pepdite 70 g Maxipro and 75 gLiquigen per L of water all products kindly donated byNutri-cia Alleroslashd Denmark) Bovine colostrumwas collected fromthe first milking after parturition The products were storedat minus20∘C and warmed to body temperature in a water bathbefore feeding to the pigs (feeding dose interval 15mLkgbody weight3 h) Colostrum was diluted in tap water toobtain the same dry matter content as in the milk formulabefore use All pigs were euthanized within the first 50 h afterinitiation of enteral nutrition according to earlier protocols[16] and tissue samples from the distal small intestinalregion were immediately snap-frozen in liquid nitrogen andstored at minus80∘C All animal protocols and procedures wereapproved by the Danish National Committee on AnimalExperimentation

23 Gene Expression Analysis Total RNA from IPEC-J2 cellswas extracted using RNeasy Mini kit (Qiagen BallerupDenmark) and on-column DNAse treated using RNase-freeDNase set (Qiagen) according to manufacturerrsquos protocolDistal intestinal tissue was homogenized and total RNAwas extracted using RNeasy Lipid Tissue Midi kit (Qiagen)and on-column DNAse treated using RNase-free DNase set(Qiagen) according to the manufacturerrsquos protocol Purityof extracted total RNA was assessed using UV absorptionspectrums including OD 260280 and OD 260230 ratios ona Nanodrop ND-1000 spectrophotometer (Saveen BiotechAarhus Denmark) and total RNA was quantified at OD 260Quality (integrity) of extracted total RNA was determinedusing on-chip electrophoresis on an Agilent 2100 Bioanalyzer(Agilent TechnologiesNaeligrumDenmark) AnRNA integritynumber was assigned to each sample using the 2100 Expertsoftware (Agilent Technologies vB0201)

Extracted RNAwas converted into cDNAby reverse tran-scription of 500 ng total RNA using the QuantiTECTReverseTranscription kit (Qiagen) containing a mix of random pri-mers and oligo-dT according to the manufacturerrsquos instruc-tions cDNAwas diluted 1 7 in lowEDTATE-buffer (VWRmdashBie amp Berntsen Herlev Denmark) prior to pre amplificationPre amplification was performed using TaqMan PreAmpMaster Mix (Applied Biosystems Naeligrum Denmark) Stocksof 200 nM primer mix were prepared combining equal con-centration of all primers used in the present study (Table 1)and in the following the genes will be denoted by theirgene symbol (Table 1) TaqMan PreAmp Master Mix (5120583L)was mixed with 25120583L 200 nM stock primer mix and 25120583Ldiluted cDNA and incubated at 95∘C in 10min followed by16 cycles of 95∘C in 15 sec and 60∘C in 4min Pre amplifiedcDNA was diluted at least 1 4 in low EDTA TE-buffer(VWRmdashBie amp Berntsen) before qPCR

Primers were designed using Primer3 (httpfrodowimitedu) as described in [17] and synthesized at TAG

ISRN Genomics 3

Table 1 Gene symbol forward and reverse primer sequences andamplicon length

Genesymbol Sequence (51015840-31015840) Amplicon

length

ACTB F CTACGTCGCCCTGGACTTC 76R GCAGCTCGTAGCTCTTCTCC

ALPI F TCCCAGACATACAACGTGGA 90R GGTCTGGTAGTTGGCCTTGA

AOAH F GTAATGGCATTTGGGGTGTC 97R TCTCCCAGCAAAATGATTCC

APOA1 F GTTCTGGGACAACCTGGAAA 86R GCTGCACCTTCTTCTTCACC

CCL2 F GCAAGTGTCCTAAAGAAGCAGTG 103R TCCAGGTGGCTTATGGAGTC

CCL3 F CCAGGTCTTCTCTGCACCAC 90R GCTACGAATTTGCGAGGAAG

CCL5 F CTCCATGGCAGCAGTCGT 121R AAGGCTTCCTCCATCCTAGC

CD14 F GGGTTCCTGCTCAGATTCTG 164R CCCACGACACATTACGGAGT

CD40 F TGAGAGCCCTGGTGGTTATC 90R GCTCCTTGGTCACCTTTCTG

CD163 F CACATGTGCCAACAAAATAAGAC 130R CACCACCTGAGCATCTTCAA

CD200 F TCCCCAGGAAGTTTTGATTG 84R CCATGGTTCTTGCTGAAGGT

CLDN3 F ATCGGCAGCAGCATTATCAC 94R ACACTTTGCACTGCATCTGG

CRP F GGTGGGAGACATTGGAGATG 85R GAAGGTCCCACCAGCATAGA

CXCL10 F CCCACATGTTGAGATCATTGC 141R GCTTCTCTCTGTGTTCGAGGA

C3 F ATCAAATCAGGCTCCGATGA 76R GGGCTTCTCTGCATTTGATG

DEFB1 F ACCTGTGCCAGGTCTACTAAAAA 109R GGTGCCGATCTGTTTCATCT

DEFB4A F CAGGATTGAAGGGACCTGTT 99R CTTCACTTGGCCTGTGTGTC

FGG F GAATTTTGGCTGGGAAATGA 86R CAGTCCTCCAGCTGCACTCT

HP F ACAGATGCCACAGATGACAGC 105R CGTGCGCAGTTTGTAGTAGG

HPRT1 F ACACTGGCAAAACAATGCAA 71R TGCAACCTTGACCATCTTTG

IL1B F CCAAAGAGGGACATGGAGAA 123R GGGCTTTTGTTCTGCTTGAG

IL1RN F TGCCTGTCCTGTGTCAAGTC 90R GTCCTGCTCGCTGTTCTTTC

Table 1 Continued


length

IL6 F TGGGTTCAATCAGGAGACCT 116R CAGCCTCGACATTTCCCTTA

IL8 F TTGCCAGAGAAATCACAGGA 78R TGCATGGGACACTGGAAATA

IL10 F CTGCCTCCCACTTTCTCTTG 95R TCAAAGGGGCTCCCTAGTTT

IL18 F CTGCTGAACCGGAAGACAAT 100R TCCGATTCCAGGTCTTCATC

IFNG F CCATTCAAAGGAGCATGGAT 76R TTCAGTTTCCCAGAGCTACCA

ITIH41 F ATGACAGCAAGCGAACAGTG 85R GGGGATCCCTCTTGGTAATC

ITIH42 F AGGCCCTCACCATATCACAG 110R GTTGCCATCCAGGACTGTTT

LBP F CCCAAGGTCAATGATAAGTTGG 83R ATCTGGAGAACAGGGTCGTG

LCT F CACTCAAAGCTGTGCAGGAC 144R GGATCCTTGGCAGAGAAGTG

MUC1 F GGATTTCTGAATTGTTTTTGCAG 116R ACTGTCTTGGAAGGCCAGAA

MUC2 F GCACGTCTGCAACAAGGAC 125R CAAAGCCCTCCAGGCAGT

NFKBIA F GAGGATGAGCTGCCCTATGAC 85R CCATGGTCTTTTAGACACTTTCC

NFKB1 F CTCGCACAAGGAGACATGAA 97R GGGTAGCCCAGTTTTTGTCA

OCLN F CGGTGAGAAGATTGGCTGAT 100R TTTCAAAAGGCCTGGATGAC

PAFAH1B1 F GCAAACTGGCTACTGTGTGAAG 113R GCACAGTCTGGTCATTGGAA

PTGS2 F AGGCTGATACTGATAGGAGAAACG 100R GCAGCTCTGGGTCAAACTTC

RPL13A F ATTGTGGCCAAGCAGGTACT 76R AATTGCCAGAAATGTTGATGC

SAA F TAAAGTGATCAGCAATGCCAAA 96R TCAACCCTTGAGTCCTCCAC

SFTPA1 F CATGGGTGTCCTCAGTTTCC 86R CATCAAAAGCGACTGACTGC

SLC5A1 F CTGCAAGAGAGTCAATGAGGAG 99R CCGGTTCCATAGGCAAACT

TF F CTCAACCTCAAAACTCCTGGAA 82R CCGTCTCCATCAGGTGGTA

TGFB1 F GCAAGGTCCTGGCTCTGTA 97R TAGTACACGATGGGCAGTGG

4 ISRN Genomics

Table 1 Continued


length

TLR2 F GTTTTACGGAAATTGTGAAACTG 136R TCCACATTACCGAGGGATTT

TLR4 F TTTCCACAAAAGTCGGAAGG 145R CAACTTCTGCAGGACGATGA

TNF F CCCCCAGAAGGAAGAGTTTC 92R CGGGCTTATCTGAGGTTTGA

TNFAIP3 F CCCAGCTTTCTCTCATGGAC 113R TTGGTTCTTCTGCCGTCTCT

Copenhagen (Copenhagen Denmark) Primer sequencesand amplicon length are shown in Table 1 The 48 genes werechosen based on a previous study showing expression ofthe selected reference- epithelial- and immune-related genesin neonatal pig tissue [18] Primer amplification efficienciesand dynamic range were acquired from standard curves con-structed from dilution series of highly responding samplesMelting curves were inspected for all primer assays andagarose gel electrophoresis and sequencing of most ampli-cons were performed to ensure primer specificity

Quantitative PCR (qPCR) was performed in 4848 Dyna-mic Array integrated fluidic circuits (Fluidigm CorporationSan Francisco CA USA) combining 48 samples with 48primer sets for 2304 simultaneous qPCR reactions Reactionmix was prepared using the following components for eachof the 48 samples 3 120583L ABI TaqMan Gene ExpressionMasterMix (Applied Biosystems) 03 120583L 20X DNA binding dyesample loading reagent (Fluidigm Corporation) 03120583L 20XEvaGreen (Biotium VWRmdashBie amp Berntsen) and 09 120583L lowEDTA TE buffer (VWRmdashBie amp Berntsen) Reaction mix(45 120583L) was mixed with 15120583L preamplified cDNA Primermix for each of the 48 primer sets was prepared using23 120583L 20120583M specific primer (forward and reverse) 25120583L2X assay loading reagent (Fluidigm Corporation) and 02 120583Llow EDTA TE buffer (VWRmdashBie amp Berntsen) Reaction mixincluding cDNA (6120583L) and primermix (5120583L) was dispensedand loaded into the integrated fluidic circuit of the DynamicArray using the IFC Controller (Fluidigm Corporation)After loading the Dynamic Array was placed in the BioMarkreal-time PCR instrument (Fluidigm Corporation) and thefollowing cycle parameter was used 2min at 50∘C 10minat 95∘C followed by 35 cycles with denaturing for 15 s at95∘C and annealingelongation for 1min at 60∘C Meltingcurves were generated to confirm a single-PCR productfor each reaction (from 60∘C to 95∘C increasing 1∘C3 s)Reactions were performed in duplicates (cDNA replicates)Non-template controls were included to indicate potentialproblems with nonspecific amplification or sample contam-inations Quantification cycle (119862119902) was acquired using thefluidigm real-time PCR analysis software 302 (FluidigmCorporation) and exported to GenEx5 (MultiD Analyses ABGoteborg Sweden)

lt415473588703818933

104811631278139315081623

173818531968208321982313

2428gt2485gt3500

PIG-COLOSPIG-COLOSPIG-COLOS

PIG-FORMPIG-FORMPIG-FORM

NTCCELL-COLOSCELL-COLOSCELL-COLOSCELL-FORMCELL-FORMCELL-FORM

ITIH

4MUC2

LPB

LCT

ALPI

CD163

CCL2

CCL3

AOAH

MUC1

DEF

B1OCL

NIL1R

NIL6

TNFA

IP3

SLC5

A1TL

R4

DEF

B4A

PAFA

H1B

1NFK

B1IL8

IL18

NFK

BIA

CXCL

10CL

DN3

CD14

CCL5

HPR

T1RP

L13A

ACTB

TGFB

1

Figure 1 Heat map showing the 119862119902 value of non-normalized rawdata from representative distal small intestinal samples of pretermpigs and IPEC-J2 cells in addition to a non-template control (NTC)Ahigh gene expression corresponds to a low119862119902 value (yellow)whilea low gene expression corresponds to a high119862119902 value (purpleblack)Relative quantities of differentially expressed genes can be seen inFigure 2

24 Data Analysis and Statistics Data preprocessing nor-malization relative quantification and statistics were per-formed using GenEx5 (MultiD Analyses AB) Data were pre-processed as follows (1) Data were corrected for PCR effi-ciency for each primer assay individually (2) Interdynamicarray variation was compensated by using several highlystable samples as interdynamic array calibrators (3) Hypox-anthine phosphoribosyltransferase 1 (HPRT1) and beta-actin(ACTB) were found to be the most stably expressed referencegenes in the present study using both GeNorm [19] andNormFinder [20] therefore the geometric mean of thesetwo genes was used to normalize all samples in GenEx5 (4)The average of cDNA technical repeats was performed afterreference gene normalization but before 119862119902 was transformedto linear scale (relative quantities) In rare cases of highstandard deviation between the two cDNA replicates oneor both of the samples or the primer assay were excludedfrom the analysis based on visual inspection of fluorescenceand melting curves To visualize differential gene expressionrelative expression for all samples was calculated relative tothe sample with the lowest expression for each primer set inthe group of samples tested Data were log

2transformed prior

to 119905-test ANOVA and principal component analysis (PCA)Gene expression was considered significantly different if the119875 value lt005 and the relative expression gt20

3 Results

An overview of raw data for genes expressed in intestinaltissue from preterm pigs and in IPEC-J2 cells is presentedin the heat map (Figure 1) based on color coding of theexpression level before preprocessing After preprocessing

ISRN Genomics 5

Gene expression in preterm pig intestine

PIG-COLOSPIG-FORM

40

30

20

10

0

Relat

ive e

xpre

ssio

n

a

a a aab

b

b

ab

DEFB1 CXCL10 IL1RN ALPI IL8

(a)

Gene expression in IPEC-J2 cells

CELL-COLOSCELL-CONTR

CELL-FORM

8

6

4

2

0

a a a

a

a

a

aa a

a

abb


Relat

ive e

xpre

ssio

n

(b)

Figure 2 Relative expression (mean plusmn SEM) of genes expressed significantly different in intestinal tissue of PIG-COLOS versus PIG-FORM(a) and between different IPEC-J2 cell treatment groups CELL-CONTR CELL-COLOS and CELL-FORM (b) Relative expression for allsamples was calculated relative to the sample with the lowest expression within each gene in the group of samples tested Different superscriptletters within each gene indicate significant difference (119875 lt 005)

including visual inspection of melting curves 31 reference-and epithelial- and immune response-related genes wereexpressed within the dynamic range in pig intestinal tissueand of these 22 genes were expressed in IPEC-J2 cells Nogenes were expressed only in IPEC-J2 cells

In PIG-COLOSDEFB1wasmore highly expressed whileCXCL10 IL1RN and ALPI were less expressed than in PIG-FORM intestinal tissue (Figure 2(a)) In IPEC-J2 cells nodifferences were seen in the expression of DEFB1 CXCL10and IL1RN between the three groups and ALPI was notexpressed in IPEC-J2 cells In contrast IL8 expression wassignificantly higher in CELL-FORM compared with CELL-CONTR with intermediate expression in CELL-COLOS(Figure 2(b)) When comparing CELL-FORM and CELL-COLOS with PIG-FORM and PIG-COLOS in a PCA twomajor groups were identified (Figure 3) discriminating geneexpression profiles of pig intestinal tissue from that of IPEC-J2 cells Furthermore discrimination between PIG-COLOSand PIG-FORM was achieved by PCA while no discrimina-tion between CELL-COLOS andCELL-FORMwas observedAs expected major differences in gene expression wereseen between IPEC-J2 cells and pig intestinal tissue whichaccounted for the clear clustering in the PCA

Transcript coding for CXCL10 CCL5 and SLC5A1 wasfound at a higher level in pig intestinal tissue compared withIPEC-J2 cells in contrast the expression of CLDN3 DEFB1IL1RN IL6 MUC1 and OCLN was lower in intestinal tissuecompared with IPEC-J2 cells (data not shown)

4 Discussion

In this study the IPEC-J2 cell line was evaluated as a possiblein vitro model for investigation of the gene expression of

3210minus1

minus2

minus3

minus4

minus5

minus12 minus10 minus8 minus6 minus4 minus2 0 2 4 6PC1

PC2

Figure 3 Principal component analysis of expression data fromdistal small intestinal tissue from preterm pigs PIG-FORM (whitecircles) and PIG-COLOS (black circles) and IPEC-J2 cells CELL-FORM (white squares) and CELL-COLOS (black squares)

IECs in relation to dietary effects on the neonatal intestineTo our knowledge this is the first demonstration of CCL5CD14 CXCL10 IL1RN PAFAH1B1 and SLC5A1 expressionin IPEC-J2 cells The expression of DEFB1 DEFB4A IL6IL8 IL18 MUC1 NFKBIA NFKB1 OCLN TLR4 TGFB1and TNFAIP3 has been reported previously [2ndash6 21] On theother hand a lack ofTGFB1 expression has also been reportedin infection studies [2] Finally we confirmed the lack ofexpression of CCL2 [4] andMUC2 [2] in IPEC-J2 cells

We found that the IPEC-J2 cells and intestinal tissueclustered in two distinct groups in the PCAof gene expressionpatterns Furthermore it was possible by PCA to discriminatebetween the gene expression profiles of intestinal tissue from

6 ISRN Genomics

preterm pigs in different diet groups however discrimina-tion by PCA between the IPEC-J2 cell diet groups CELL-FORM and CELL-COLOS was not possible Four geneswere differentially expressed between pigs fed milk formulaor colostrum DEFB1 encoding the antimicrobial proteindefensin beta1 [22] was upregulated in PIG-COLOS com-pared with PIG-FORM which suggests that colostrum maystimulate the host antimicrobial response The expression ofALPI coding for the enzyme intestinal alkaline phosphatasein addition to CXCL10 and IL1RN was downregulated inPIG-COLOS comparedwith PIG-FORMCXCL10 is involvedin T-lymphocyte activation and induction of chemotaxistoward infected tissues [23] and IL1RN coding for the IL-1 receptor antagonist has previously been found to showsimilar expression patterns as CXCL10 in viral lung infectionof pigs [24] None of these four genes were differentiallyexpressed in IPEC-J2 cells However the expression of IL8coding for the potent neutrophil chemoattractant IL-8 washigher in CELL-FORM relative to CELL-CONTRThusmilkformula might induce specific parts of a proinflammatoryresponse in IPEC-J2 cells Of the genes up-regulated in IPEC-J2 cells relative to pig intestinal tissue MUC1 and DEFB1stood out as highly differentially expressed with a relativeexpression of more than 1000 (data not shown) These genesare coding for proteins secreted by IECs and involved in thefirst line of defense and thus expected to be highly expressedin the IPEC-J2 cell line which consist of IECs only in contrastto the intestinal tissue consisting of many different cell types

The IPEC-J2 cell line may under the conditions usedin this study not be an optimal model to investigate diet-related effects since the gene expression differences foundin intestinal tissue could not be demonstrated in the IPEC-J2 cell line The expression differences between IPEC-J2 cellsand preterm pig intestinal tissue are possibly due to the hete-rogeneous population of cells in intestinal tissue in additionto the intestinal microbiota which may affect the expressionlevel of certain genes On the other hand the IPEC-J2 cellline is a homogenous cell population consisting of IECs onlyIn vivo digestion of the diet may also change its effect onIECs and exposure to artificially digested diets of the IPEC-J2 cells may provide a situation that more closely resemblesthat of the intestine Furthermore studies could include geneexpression analysis of intestinal samples from term bornpigs to further determine if the observed differences in geneexpression could be due to the difference in maturationalstate

5 Conclusion

The observed differences in the diet-dependent gene expres-sion patterns between IPEC-J2 cells and intestinal tissue frompreterm newborn pigs underline that responses to envi-ronmental stimuli (eg diet) may differ markedly betweenisolated enterocyte cell systems and intact tissue responsesboth acting as models for the sensitive newborn intestineHowever this in vitro cellmodel still provides the opportunityto investigate the interaction between a limited number offactors in a standardized setting although caution must be

exercised in the interpretation of diet-induced effects on geneexpression in this model

Conflict of Interests

The authors claim to have no conflict of interests in the con-text of this work

Acknowledgments

The authors wish to thank Henriette Vorsholt and Karin TarpWendt at Innate Immunology Group The National Veteri-nary Institute Technical University of Denmark for skilledtechnical assistance with RNA extraction and qPCR analysis

References

[1] R S Pitman and R S Blumberg ldquoFirst line of defense therole of the intestinal epithelium as an active component of themucosal immune systemrdquo Journal of Gastroenterology vol 35no 11 pp 805ndash814 2000

[2] P SchierackMNordhoffM Pollmann et al ldquoCharacterizationof a porcine intestinal epithelial cell line for in vitro studies ofmicrobial pathogenesis in swinerdquo Histochemistry and Cell Biol-ogy vol 125 no 3 pp 293ndash305 2006

[3] E J A Veldhuizen I Koomen T Ultee A van Dijk and H PHaagsman ldquoSalmonella serovar specific upregulation of porcinedefensins 1 and 2 in a jejunal epithelial cell linerdquo VeterinaryMicrobiology vol 136 no 1-2 pp 69ndash75 2009

[4] VMariani S Palermo S Fiorentini A Lanubile andEGiuffraldquoGene expression study of two widely used pig intestinalepithelial cell lines IPEC-J2 and IPI-2IrdquoVeterinary Immunologyand Immunopathology vol 131 no 3-4 pp 278ndash284 2009

[5] T E Burkey K A Skjolaas S S Dritz and J E MintonldquoExpression of porcine Toll-like receptor 2 4 and 9 gene tran-scripts in the presence of lipopolysaccharide and Salmonellaenterica serovars Typhimurium and Choleraesuisrdquo VeterinaryImmunology and Immunopathology vol 130 no 1-2 pp 96ndash1012009

[6] M M Geens and T A Niewold ldquoPreliminary characterizationof the transcriptional response of the porcine intestinal cell lineIPEC-J2 to enterotoxigenic escherichia coli escherichia coliand E coli lipopolysacchariderdquo Comparative and FunctionalGenomics vol 2010 Article ID 469583 2010

[7] L D Schmidt L J Kohrt and D R Brown ldquoComparisonof growth phase on Salmonella enterica serovar Typhimuriuminvasion in an epithelial cell line (IPEC J2) andmucosal explantsfrom porcine small intestinerdquoComparative Immunology Micro-biology and Infectious Diseases vol 31 no 1 pp 63ndash69 2008

[8] AK SonntagM Bielaszewska AMellmann et al ldquoShiga toxin2e-producing Escherichia coli isolates from humans and pigsdiffer in their virulence profiles and interactions with intestinalepithelial cellsrdquo Applied and Environmental Microbiology vol71 no 12 pp 8855ndash8863 2005

[9] P W Lin and B J Stoll ldquoNecrotising enterocolitisrdquoThe Lancetvol 368 no 9543 pp 1271ndash1283 2006

[10] M A Quigley G Henderson M Y Anthony andW McGuireldquoFormula milk versus donor breast milk for feeding pretermor low birth weight infantsrdquo Cochrane Database of SystematicReviews no 4 Article ID CD002971 2007

ISRN Genomics 7

[11] C R Bjornvad TThymann N E Deutz et al ldquoEnteral feedinginduces diet-dependent mucosal dysfunction bacterial prolif-eration and necrotizing enterocolitis in preterm pigs on paren-teral nutritionrdquo American Journal of Physiology vol 295 no 5pp G1092ndashG1103 2008

[12] P T Sangild R H Siggers M Schmidt et al ldquoDiet- andcolonization-dependent intestinal dysfunction predisposes tonecrotizing enterocolitis in preterm pigsrdquoGastroenterology vol130 no 6 pp 1776ndash1792 2006

[13] T Thymann D G Burrin K A Tappenden C R Bjornvad SK Jensen and P T Sangild ldquoFormula-feeding reduces lactosedigestive capacity in neonatal pigsrdquo British Journal of Nutritionvol 95 no 6 pp 1075ndash1081 2006

[14] E R Van Haver P T Sangild M Oste J L A Siggers A LM Weyns and C J Van Ginneken ldquoDiet-dependent mucosalcolonization and interleukin-1120573 responses in preterm pigssusceptible to necrotizing enterocolitisrdquo Journal of PediatricGastroenterology and Nutrition vol 49 no 1 pp 90ndash98 2009

[15] R J Playford C E Macdonald andW S Johnson ldquoColostrumand milk-derived peptide growth factors for the treatment ofgastrointestinal disordersrdquo American Journal of Clinical Nutri-tion vol 72 no 1 pp 5ndash14 2000

[16] R H Siggers TThymann B B Jensen et al ldquoElective cesareandelivery affects gut maturation and delays microbial coloniza-tion but does not increase necrotizing enterocolitis in pretermpigsrdquo American Journal of Physiology vol 294 no 3 pp R929ndashR938 2008

[17] K Skovgaard S Mortensen M Boye J Hedegaard and PM H Heegaard ldquoHepatic gene expression changes in pigsexperimentally infected with the lung pathogen Actinobacil-lus pleuropneumoniae as analysed with an innate immunityfocused microarrayrdquo Innate Immunity vol 16 no 6 pp 343ndash353 2010

[18] M S Cilieborg K Skovgaard L M Noslashrgaard P T Sangildand M Boye ldquoIntestinal colonization gut function and inflam-matory responses are moderately influenced by gestational ageat birthrdquo in Proceedings of the 14th International Symposium onMicrobial Ecology 2012

[19] J Vandesompele K De Preter F Pattyn et al ldquoAccurate nor-malization of real-time quantitative RT-PCR data by geometricaveraging of multiple internal control genesrdquo Genome Biologyvol 3 no 7 Article ID RESEARCH0034 2002

[20] C L Andersen J L Jensen and T F Oslashrntoft ldquoNormalization ofreal-time quantitative reverse transcription-PCR data a model-based variance estimation approach to identify genes suited fornormalization applied to bladder and colon cancer data setsrdquoCancer Research vol 64 no 15 pp 5245ndash5250 2004

[21] H R Sargeant H M Miller and M A Shaw ldquoInflammatoryresponse of porcine epithelial IPEC J2 cells to enterotoxigenic Ecoli infection is modulated by zinc supplementationrdquoMolecularImmunology vol 48 pp 2113ndash2121 2011

[22] R I Lehrer ldquoPrimate defensinsrdquo Nature Reviews Microbiologyvol 2 no 9 pp 727ndash738 2004

[23] A M E Sidahmed A J Leon S E Bosinger et al ldquoCXCL10contributes to p38-mediated apoptosis in primary T lympho-cytes in vitrordquo Cytokine vol 59 no 2 pp 433ndash441 2012

[24] K Skovgaard S Cirera D Vasby et al ldquoExpression of innateimmune genes proteins and microRNAs in lung tissue of pigsinfected experimentally with influenza virus (H1N2)rdquo InnateImmunity 2013

Submit your manuscripts athttpwwwhindawicom

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

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International Journal of

Volume 2014

Zoology


Molecular Biology International

Hindawi Publishing Corporationhttpwwwhindawicom

GenomicsInternational Journal of

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The Scientific World JournalHindawi Publishing Corporation httpwwwhindawicom Volume 2014


BioinformaticsAdvances in

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Signal TransductionJournal of

BioMed Research International


Evolutionary BiologyInternational Journal of



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Genetics Research International


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Volume 2014

Stem CellsInternational



Enzyme Research



Microbiology

2 ISRN Genomics

Compared with animal models cell line studies are lesscost intensive associated with no ethical concerns and pro-vide a highly-controlled simple model to investigate isolatedfactors for example diet on the IECs response The IPEC-J2 cell line has already been shown to be a valuable model forthe investigation of host-pathogen interactions and could alsobe a promising model for in vitro studies of innate immunefunctions of neonatal IECs in response to dietary stimuliIn this study we evaluated the potential of the IPEC-J2 cellline as an in vitro model to study diet-induced effects on thepreterm neonatal intestine We analyzed and compared theexpression of epithelial- and immune response-related genesin the IPEC-J2 cell line and in preterm pig intestinal tissue

2 Materials and Methods

21 IPEC-J2 Cell Line Study IPEC-J2 cells [2] were main-tained inDulbeccorsquosmodified eaglemedium (DMEM)HamrsquosF-12 (1 1) supplemented with 10 fetal calf serum penicillin(100UmL) streptomycin (100 120583gmL) pyruvate (1mM)and L-glutamine (2mM) (growth medium Sigma-AldrichBroslashndbyDenmark) in a humidified atmosphere of 5CO

2at

37∘C Passage was performed approximately once a week andcells between passages 82ndash85 were used in the experimentPrior to the experiment the cell culture was found to be freeof mycoplasma

For the experiment cells were transferred to Corn-ing Transwell-COL collagen coated-membrane (04 120583m poresize Sigma-Aldrich) and grown in growth medium sup-plemented with epidermal growth factor (5mgmL SigmaAldrich) and insulin-transferrin-selenium (5 120583gmL of eachSigma-Aldrich) until stable transepithelial electric resistancevalues measured with an EVOMmdashEpithelial Voltohmme-ter (World Precision Instruments Berlin Germany) werereached after approximately 12 d The cells were stimulatedfor 2 h in three different diet solutions growthmedium alone(CELL-CONTR 119899 = 4) growth medium containing 1gamma-irradiated bovine colostrum (CELL-COLOS 119899 =4) or growth medium containing 1 milk formula (CELL-FORM 119899 = 4) These conditions were selected based onresults from preexperiments testing cell viability under dif-ferent concentration of colostrum and periods of time Thecolostrum and milk formula were identical to those used inthe pig study (see later) The lipid fraction and cellular debriswere removed by centrifugation at 3500 rpm for 20min at4∘C After stimulation the cells were gently washed twicewith Dulbeccorsquos phosphate buffered saline (D8537 Sigma-Aldrich) harvested and stored at minus80∘C

22 Preterm Pig Study Nineteen preterm pigs were deliveredfrom four sows by Caesarean section (Large White times DanishLandracetimesDuroc Askelygaard Farm Roskilde Denmark) at105ndash107 d of gestation (90ndash92 gestation) Procedures forCaesarean section and nursing of the preterm pigs followed astandard protocol previously described in details [11 12] Thepigs were given total parenteral nutrition through a vascularcatheter (4mLkgh advancing to 6mLkgh) for 2 d basedon Nutriflex Lipid Plus (Braun Melsungen Germany) and

adjusted in nutrient composition to meet the requirementsof pigs as described by [11] Hereafter pigs were randomizedaccording to birth weight into two enteral nutrition groupsgamma-irradiated (1 times 10 kGy Sterigenics Espergaeligrde Den-mark) bovine colostrum (PIG-COLOS 119899 = 6 kindly donatedby Biofiber-Damino AS Gesten Denmark) or milk formula(PIG-FORM 119899 = 13 80 g Pepdite 70 g Maxipro and 75 gLiquigen per L of water all products kindly donated byNutri-cia Alleroslashd Denmark) Bovine colostrumwas collected fromthe first milking after parturition The products were storedat minus20∘C and warmed to body temperature in a water bathbefore feeding to the pigs (feeding dose interval 15mLkgbody weight3 h) Colostrum was diluted in tap water toobtain the same dry matter content as in the milk formulabefore use All pigs were euthanized within the first 50 h afterinitiation of enteral nutrition according to earlier protocols[16] and tissue samples from the distal small intestinalregion were immediately snap-frozen in liquid nitrogen andstored at minus80∘C All animal protocols and procedures wereapproved by the Danish National Committee on AnimalExperimentation

23 Gene Expression Analysis Total RNA from IPEC-J2 cellswas extracted using RNeasy Mini kit (Qiagen BallerupDenmark) and on-column DNAse treated using RNase-freeDNase set (Qiagen) according to manufacturerrsquos protocolDistal intestinal tissue was homogenized and total RNAwas extracted using RNeasy Lipid Tissue Midi kit (Qiagen)and on-column DNAse treated using RNase-free DNase set(Qiagen) according to the manufacturerrsquos protocol Purityof extracted total RNA was assessed using UV absorptionspectrums including OD 260280 and OD 260230 ratios ona Nanodrop ND-1000 spectrophotometer (Saveen BiotechAarhus Denmark) and total RNA was quantified at OD 260Quality (integrity) of extracted total RNA was determinedusing on-chip electrophoresis on an Agilent 2100 Bioanalyzer(Agilent TechnologiesNaeligrumDenmark) AnRNA integritynumber was assigned to each sample using the 2100 Expertsoftware (Agilent Technologies vB0201)

Extracted RNAwas converted into cDNAby reverse tran-scription of 500 ng total RNA using the QuantiTECTReverseTranscription kit (Qiagen) containing a mix of random pri-mers and oligo-dT according to the manufacturerrsquos instruc-tions cDNAwas diluted 1 7 in lowEDTATE-buffer (VWRmdashBie amp Berntsen Herlev Denmark) prior to pre amplificationPre amplification was performed using TaqMan PreAmpMaster Mix (Applied Biosystems Naeligrum Denmark) Stocksof 200 nM primer mix were prepared combining equal con-centration of all primers used in the present study (Table 1)and in the following the genes will be denoted by theirgene symbol (Table 1) TaqMan PreAmp Master Mix (5120583L)was mixed with 25120583L 200 nM stock primer mix and 25120583Ldiluted cDNA and incubated at 95∘C in 10min followed by16 cycles of 95∘C in 15 sec and 60∘C in 4min Pre amplifiedcDNA was diluted at least 1 4 in low EDTA TE-buffer(VWRmdashBie amp Berntsen) before qPCR

Primers were designed using Primer3 (httpfrodowimitedu) as described in [17] and synthesized at TAG

ISRN Genomics 3



length























Table 1 Continued


length























4 ISRN Genomics

Table 1 Continued


length







lt415473588703818933

104811631278139315081623

173818531968208321982313

2428gt2485gt3500




ITIH

4MUC2

LPB

LCT

ALPI

CD163

CCL2

CCL3

AOAH

MUC1

DEF

B1OCL

NIL1R

NIL6

TNFA

IP3

SLC5

A1TL

R4

DEF

B4A

PAFA

H1B

1NFK

B1IL8

IL18

NFK

BIA

CXCL

10CL

DN3

CD14

CCL5

HPR

T1RP

L13A

ACTB

TGFB

1



2transformed prior


3 Results


ISRN Genomics 5


PIG-COLOSPIG-FORM

40

30

20

10

0

Relat

ive e

xpre

ssio

n

a

a a aab

b

b

ab


(a)



CELL-FORM

8

6

4

2

0

a a a

a

a

a

aa a

a

abb


Relat

ive e

xpre

ssio

n

(b)





4 Discussion


3210minus1

minus2

minus3

minus4

minus5


PC2




6 ISRN Genomics



5 Conclusion





Acknowledgments


References











ISRN Genomics 7






















Volume 2014

Zoology





Volume 2014


















Advances in

Virolog y



Volume 2014




Enzyme Research



Microbiology

ISRN Genomics 3



length























Table 1 Continued


length























4 ISRN Genomics

Table 1 Continued


length







lt415473588703818933

104811631278139315081623

173818531968208321982313

2428gt2485gt3500




ITIH

4MUC2

LPB

LCT

ALPI

CD163

CCL2

CCL3

AOAH

MUC1

DEF

B1OCL

NIL1R

NIL6

TNFA

IP3

SLC5

A1TL

R4

DEF

B4A

PAFA

H1B

1NFK

B1IL8

IL18

NFK

BIA

CXCL

10CL

DN3

CD14

CCL5

HPR

T1RP

L13A

ACTB

TGFB

1



2transformed prior


3 Results


ISRN Genomics 5


PIG-COLOSPIG-FORM

40

30

20

10

0

Relat

ive e

xpre

ssio

n

a

a a aab

b

b

ab


(a)



CELL-FORM

8

6

4

2

0

a a a

a

a

a

aa a

a

abb


Relat

ive e

xpre

ssio

n

(b)





4 Discussion


3210minus1

minus2

minus3

minus4

minus5


PC2




6 ISRN Genomics



5 Conclusion





Acknowledgments


References











ISRN Genomics 7






















Volume 2014

Zoology





Volume 2014


















Advances in

Virolog y



Volume 2014




Enzyme Research



Microbiology

4 ISRN Genomics

Table 1 Continued


length







lt415473588703818933

104811631278139315081623

173818531968208321982313

2428gt2485gt3500




ITIH

4MUC2

LPB

LCT

ALPI

CD163

CCL2

CCL3

AOAH

MUC1

DEF

B1OCL

NIL1R

NIL6

TNFA

IP3

SLC5

A1TL

R4

DEF

B4A

PAFA

H1B

1NFK

B1IL8

IL18

NFK

BIA

CXCL

10CL

DN3

CD14

CCL5

HPR

T1RP

L13A

ACTB

TGFB

1



2transformed prior


3 Results


ISRN Genomics 5


PIG-COLOSPIG-FORM

40

30

20

10

0

Relat

ive e

xpre

ssio

n

a

a a aab

b

b

ab


(a)



CELL-FORM

8

6

4

2

0

a a a

a

a

a

aa a

a

abb


Relat

ive e

xpre

ssio

n

(b)





4 Discussion


3210minus1

minus2

minus3

minus4

minus5


PC2




6 ISRN Genomics



5 Conclusion





Acknowledgments


References











ISRN Genomics 7






















Volume 2014

Zoology





Volume 2014


















Advances in

Virolog y



Volume 2014




Enzyme Research



Microbiology

ISRN Genomics 5


PIG-COLOSPIG-FORM

40

30

20

10

0

Relat

ive e

xpre

ssio

n

a

a a aab

b

b

ab


(a)



CELL-FORM

8

6

4

2

0

a a a

a

a

a

aa a

a

abb


Relat

ive e

xpre

ssio

n

(b)





4 Discussion


3210minus1

minus2

minus3

minus4

minus5


PC2




6 ISRN Genomics



5 Conclusion





Acknowledgments


References











ISRN Genomics 7






















Volume 2014

Zoology





Volume 2014


















Advances in

Virolog y



Volume 2014




Enzyme Research



Microbiology

6 ISRN Genomics



5 Conclusion





Acknowledgments


References











ISRN Genomics 7






















Volume 2014

Zoology





Volume 2014


















Advances in

Virolog y



Volume 2014




Enzyme Research



Microbiology

ISRN Genomics 7






















Volume 2014

Zoology





Volume 2014


















Advances in

Virolog y



Volume 2014




Enzyme Research



Microbiology








Volume 2014

Zoology





Volume 2014


















Advances in

Virolog y



Volume 2014




Enzyme Research



Microbiology

Documents

Gene Expression Analysis of the IPEC-J2 Cell Line: A