research article enterococcus faecium ncimb 10415...

Research ArticleEnterococcus faecium NCIMB 10415 Modulates EpithelialIntegrity Heat Shock Protein and Proinflammatory CytokineResponse in Intestinal Cells

Shanti Klingspor1 Angelika Bondzio2 Holger Martens1 Joumlrg R Aschenbach1

Katharina Bratz3 Karsten Tedin4 Ralf Einspanier2 and Ulrike Lodemann1

1 Institute of Veterinary Physiology Department of Veterinary Medicine Freie Universitat BerlinOertzenweg 19b 14163 Berlin Germany2Institute of Veterinary Biochemistry Department of Veterinary Medicine Freie Universitat BerlinOertzenweg 19b 14163 Berlin Germany3Institute of Food Hygiene Department of Veterinary Medicine Freie Universitat Berlin Konigsweg 69 14163 Berlin Germany4Institute of Microbiology and Epizootics Department of Veterinary Medicine Freie Universitat BerlinRobert-von-Ostertag-Straszlige 7-13 14163 Berlin Germany

Correspondence should be addressed to Ulrike Lodemann ulrikelodemannfu-berlinde

Received 4 July 2014 Revised 25 September 2014 Accepted 26 September 2014

Academic Editor Ishak O Tekin

Copyright copy 2015 Shanti Klingspor 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

Probiotics have shown positive effects on gastrointestinal diseases they have barrier-modulating effects and change theinflammatory response towards pathogens in studies in vitro The aim of this investigation has been to examine the response ofintestinal epithelial cells to Enterococcus faecium NCIMB 10415 (E faecium) a probiotic positively affecting diarrhea incidence inpiglets and two pathogenic Escherichia coli (E coli) strains with specific focus on the probiotic modulation of the response to thepathogenic challenge Porcine (IPEC-J2) and human (Caco-2) intestinal cells were incubated without bacteria (control) with Efaecium with enteropathogenic (EPEC) or enterotoxigenic E coli (ETEC) each alone or in combination with E faecium The ETECstrain decreased transepithelial resistance (TER) and increased IL-8 mRNA and protein expression in both cell lines comparedwith control cells an effect that could be prevented by pre- and coincubation with E faecium Similar effects were observed forthe increased expression of heat shock protein 70 in Caco-2 cells When the cells were challenged by the EPEC strain no suchpattern of changes could be observed The reduced decrease in TER and the reduction of the proinflammatory and stress responseof enterocytes following pathogenic challenge indicate the protective effect of the probiotic

1 Introduction

Probiotic bacteria have been shown to have positive effectson hosts with intestinal diseases such as Clostridium difficile-Rotavirus- and Escherichia coli-induced diarrhea and also inthe prevention of antibiotic-associated diarrhea and nosoco-mial infections [1ndash5] According to the WHO probiotics aredefined as live organisms that when ingested in sufficientamounts have a beneficial effect on the overall health ofthe host [6] In animal nutrition probiotics are used as feed

additives with positive effects regarding health and growthperformance traits [7ndash9]

The probiotic Enterococcus faecium NCIMB 10415 (Efaecium) is licensed as a feed additive for sows and pigletsand has been demonstrated to reduce diarrhea incidence andseverity in weaning piglets [10 11] In human medicine Efaecium is used successfully in the treatment of acute diar-rheal diseases and in the prevention of antibiotic-associateddiarrhea [12 13] However the crosstalk between the probi-otic and other microorganisms and between the probiotic

Hindawi Publishing CorporationMediators of InflammationVolume 2015 Article ID 304149 11 pageshttpdxdoiorg1011552015304149

2 Mediators of Inflammation

and epithelial and immune cells of the intestinal wall isextremely complex [14 15] and many of the underlyingsignaling mechanisms are still only partially understood

In vivomodels of gastrointestinal infections have demon-strated a positive effect of various probiotic feed additives onthe functional barrier of the intestine [16ndash18] These resultsare supported by data from in vitro cell culture infectionmodels in which probiotic strains prevent or amelioratedamage to epithelial integrity by a pathogenic challenge [1920] Furthermore the immunological response of themucosacan be influenced by probiotic strains which modulate therelease of cytokines amongst other effects [21 22] Forexample culture supernatant of Lactobacillus plantarum 2142had a suppressive effect on the interleukin-8 (IL-8) and tumornecrosis factor-120572 (TNF-120572) levels induced by oxidative stressin IPEC-J2 cells [23]

The expression of heat shock proteins (HSPs) a set ofproteins that are involved in many regulatory pathways andthat serve as chaperones for preserving cellular proteins canbe influenced by the intestinal microbiota and by probiotics[24ndash26] Some of these HSPs such as the inducible formof HSP70 are upregulated by several noxious conditionsand can therefore be considered as cellular stress indicators[27] HSPs and cytokines in turn are able to regulatebarrier properties by influencing tight junction proteins andthe structure and function of the cytoskeleton or transportproperties of the epithelium [24 28]

In previous studies feed supplementation with E fae-cium has been demonstrated to increase the absorptive andsecretory capacity and improve barrier function of the smallintestinal epithelium of piglets [29 30] Furthermore theproinflammatory cytokine IL-1120572 which is expressed in higheramounts in Peyerrsquos patches of E faecium-treated piglets [31]increases chloride secretion in the small intestine of pigs [29]

The hypothesis of the current study is that intestinal epi-thelial cells play an important role in innate immuneresponses during enteric infections and that E faecium inturn modulates the severity of enteric infections via thealtered generation of proinflammatory cytokines and HSPsin intestinal cells Thus the aim of the present study has beento investigate the influence of the probiotic E faecium andtwo different pathogenic E coli strains on the HSP and proin-flammatory cytokine responses and the epithelial integrityof two intestinal epithelial cell lines We have further testedwhether pre- and coincubation with E faecium change theepithelial cell response to pathogenic E coli strains

2 Materials and Methods

21 Cells and Culture Conditions The cell cultivation isdescribed in detail in Lodemann et al [32] The humanepithelial intestinal cell line from colorectal adenocarcinomaCaco-2 (ATCC Catalog number HTB-37 ATCC Manas-sas USA passages 37ndash45) was used as a model for thehuman small intestine The porcine intestinal epithelial cellline (IPEC-J2 passages 73ndash79) was used as a model forthe pig small intestine This cell line was established fromthe jejunum of a newborn pig [33] and kindly provided

by Professor Dr Anthony Blikslager (North Carolina StateUniversity USA) The cells routinely tested negative formycoplasma contamination

Cells for the experiments were allowed to differentiate for14 days (IPEC-J2) or 21 days (Caco-2) On the day prior toexperiments the cells were fed with serum- and antibiotic-free media

22 Bacterial Strains Three different bacterial strains wereused for the experiments (1) the probiotic strain Enterococ-cus faecium NCIMB 10415 (cultivated from Cylactin DSMHeerlen the Netherlands) (2) the enterotoxigenic E coliIMT4818 (ETEC isolated from a two-week-old piglet withenteritis O149K91K88 (F4) and found to be positive for thepresence of virulence genes est-1a est-2 (genes coding forheat stable enterotoxins I and II) and elt-1ab (gene codingfor heat labile enterotoxin I) by the polymerase chain reaction(PCR)) and (3) the human enteropathogenicE coliE234869(EPEC serotypeO127H6 positive for the eae gene coding forthe E coli attaching-effacing factor)

The E faecium NCIMB 10415 strain was cultivated inbrain-heart infusion (BHI) broth (OXOID GmbH WeselGermany) and the E coli strains in LB medium accordingto Miller containing 10 gL tryptone (OXOID GmbH WeselGermany) 5 gL yeast extract (OXOID GmbH Wesel Ger-many) and 10 gL NaCl at a pH of 70

After overnight incubation of the cells at 37∘C sub-cultures of bacteria were grown for 3 to 4 h until mid-log phase and then centrifuged Cell pellets were washedtwice in phosphate-buffered saline (PBS Biochrom BerlinGermany) The bacteria were resuspended in antibiotic- andserum-free Caco-2 or IPEC-J2 cell culture medium to reacha concentration of asymp108 colony-forming units (CFU)mL

The optical density was measured to determine the con-centration of bacterial cellsThemeasurement was confirmedby serial dilution on agar plates The intestinal cells wereinfected with 106 bacteria per cell culture insert (112 cm2)or per well (191 cm2) corresponding to a multiplicity ofinfection (MOI) of about 10 bacteria per seeded cell Thebacteria were added to the apical pole of the cells

23 Experimental Setup and Procedure For each experimentthe cell monolayers for the real-time quantitative PCRs (RT-qPCR) for enzyme-linked immunosorbent assay (ELISA)and for the transepithelial electrical resistance (TER) mea-surements were incubated for 2 hwith the respective bacterialstrains (ETEC EPEC or E faecium) at a concentration of106 bacteria per cell culture insert or well (see Figure 1) Thecontrol cells received the equivalent amount of bacteria-freemedium Two hours after the bacterial incubation the cellswere washed twice with gentamicin-supplemented media towash out and kill the bacteria and gentamicin-supplementedmedia were added Gentamicin was added to the media ata concentration of 50120583gmL (Biochrom Berlin Germany)The cells were incubated for various periods of time withregard to the different parameters measured the exact timeperiods being given in Figure 1 under the specific headingsWhen the cells were incubated with the probiotic and either

Mediators of Inflammation 3

Addition of Ecffor coincubations

Addition of Ecf ETEC and EPEC for themono- and coincubations and medium

for the control

Sampling for qPCRTER measurement

TER measurement

Sampling for ELISA

minus2 h 2 h 4 h 6 h 8 h

Washing and addition of gentamicin

Figure 1 Timeline for the experimental setup

the ETEC or the EPEC together the cells were preincubatedwith the E faecium for 2 h and then the pathogens wereadded The cells were in contact with the pathogens for thesame amount of time as in the monoincubation with theETEC or EPEC In the following this experimental setup willbe called ldquocoincubationrdquo and the incubation timewill be givenas the time that the cells were incubated with the pathogens

A total of six independent experiments were performedfor each cell line

24 Transepithelial Electrical Resistance (TER)MeasurementsFor TER measurements the cells were seeded at a densityof 105 cells on cell culture inserts (Transwell clear polyestermembrane 12mm diameter 112 cm2 area 04 120583m pore sizeCorning BV Schiphol-Rijk the Netherlands)The inserts forIPEC-J2 cells were coated with rat tail collagen type I (ServaElectrophoresis GmbH Heidelberg Germany) TER wasmeasured by a Millicell-ERS (Electrical Resistance SystemMillipore GmbH Schwalbach Germany) TER values weremeasured every two hours and corrected for the resistance ofblank filters and for the membrane area

25 Real-Time Quantitative Polymerase Chain Reaction (RT-qPCR) For determining mRNA expression the cells wereseeded on 24-well cell culture plates (TPP Biochrom BerlinGermany) at a density of 105 cells191 cm2 Confluent cellmonolayers were incubated with the bacterial strains asdescribed in the section ldquoExperimental Setuprdquo After 4 h cellswere washed two times with PBS stored in RNAlater RNAStabilization Reagent (Qiagen GmbH Hilden Germany)and frozen at minus20∘C

The isolation of the total RNA of the harvested cellsthe assessment of the RNA quality and the cDNA synthesisare described in detail in other publications [29 32] Thesamples had to have a RNA integrity number above 7 tobe used for RT-qPCR For the synthesis of cDNA 100 ngtotal RNA for the IPEC-J2 cells and 500 ng total RNA forthe Caco-2 cells were used Primer information is given inTable 1Three reference geneswere selected for normalization(ACTB TBP andUBP for the Caco-2 cells andGAPDH TBPand YWHAZ for the IPEC-J2 cells) The primer informationfor the reference genes can be found in Lodemann et al [32]

RT-qPCR was performed and the relative amount of thetarget genes in the experimental groups was calculated by iQ5software (Bio-Rad Laboratories GmbHMunchen Germany)as described in Klingspor et al [29]

26 Enzyme-Linked Immunosorbent Assay (ELISA) For theELISA experiments the cells were seeded on 24-well cellculture plates (TPP Biochrom Berlin Germany) at a densityof 105 cells191 cm2 and incubation with bacterial strainswas as described above After 8 h the supernatants of thecells were harvested and species-specific IL-8 ELISAs wereperformed on cell culture supernatants according to themanufacturersrsquo instructions (Invitrogen ELISAKit Swine IL-8 Invitrogen Life Technologies GmbHDarmstadt Germanyfor IPEC-J2 supernatants Thermo scientific human IL-8ELISA Kit Pierce Biotechnology Rockford USA for Caco-2 supernatants) Assays were performed in duplicate

For the HSP70 ELISA protein was extracted from thecells For this purpose the Complete Lysis-M reagent set(Roche Diagnostics Deutschland GmbH Mannheim Ger-many) was used and the lysis buffer containing a mild deter-gent in bicine buffer and protease inhibitors was preparedfollowing the manufacturersrsquo instructions The cells werewashed with PBS and 200120583L lysis buffer was added to eachwell After 5min of incubation at room temperature andgentle shaking the cell lysate was collected HSP70 ELISAof the extracted protein was performed according to themanufacturersrsquo instructions (Porcine HSP70 ELISA Kit Bio-Medical Assay for IPEC-J2 cell extracts and HSP70 ELISAKit StressMARQBiosciences forCaco-2 cell extracts) Assayswere performed in duplicate

27 Statistical Analysis Statistical evaluations were carriedout by means of the IBM SPSS-Statistics program for Win-dows version 22 (International Business Machines CorpArmonk United States of America) Graphs were plottedwith SPSS and Microsoft Excel 2010 Results are given asmeans plusmn SEM119873 refers to the number of experiments

Statistical significance of differences was assessed byvariance analysisThe fixed factor was ldquotreatment of the cellsrdquo(incubationwithmedium (control)E faecium ETEC EPECETEC or EPEC in coincubation with E faecium) An overall


Table 1 Oligonucleotide primers and amplicon length of PCR products

Gene information Primer sequence Amplicon length Accession number ReferenceHSP70(heat shock protein 70 Homosapiens)

(S) 51015840-ACT GCC CTG ATC AAG CGC-31015840(AS) 51015840-CGG GTT GGT TGT CGG AGT AG-31015840 81 bp NM 005346 [34]

IL8 (CXCL8)(interleukin-8 (chemokine (C-X-Cmotif) ligand 8) Homo sapiens)

(S) 51015840-ATG ACT TCC AAG CTG GC-31015840(AS) 51015840-ACT TCT CCA CAA CCC T-31015840 274 bp NM 0005843 [35]

HSP70(heat shock protein 70 Sus scrofa)

(S) 51015840-GTG GCT CTA CCC GCA TCC C-31015840(AS) 51015840-GCA CAG CAG CAC CAT AGG C-31015840 114 bp M29506 [36]

IL8 (CXCL8)(interleukin-8 Sus scrofa)

(S) 51015840-GGC AGT TTT CCT GCT TTC T-31015840(AS) 51015840-CAG TGG GGT CCA CTC TCA AT-31015840 154 bp X61151 [37]

analysis of the data for each cell line and each parameter(TER mRNA- and protein expression) was performed A 119875value of lt005 was assumed to indicate statistically significantdifferences If a statistical significant difference occurred inthe overall analysis each timepoint (4 h 6 h 8 h)was analyzedseparately In the case of a significant difference betweengroups (treatment of the cells) the Fisher least significantdifference post hoc test was performed

3 Results

31 TER Measurements The TER values of confluent cellmonolayers were recorded as ameasure of epithelial integrity

311 Caco-2 The overall analysis revealed significant differ-ences of TER between incubations with the various bacteria(119875 lt 0001) Monoincubation of the cells with the probioticE faecium caused an increase of TER compared with thecontrol (at 4 h Figure 2) indicating a positive effect of Efaecium on barrier function Exposure of the cells to ETECsignificantly decreased TER (at 4 h and 6 h) whereas the TERof EPEC-treated cells did not show significant alterationscompared with the control Cell monolayers coincubatedwith E faecium and ETEC showed a significantly higher TERcompared with cells incubated with ETEC alone

312 IPEC-J2 In IPEC-J2 cells significant differencesbetween the various bacterial incubations could also bedetected (119875 = 001) In this cell line the TER values were notaffected in cells that were incubated with E faecium alonecompared with the control (Figure 3) However the ETECstrain again caused a significant decrease in TER (4 h 6 h)whereas the TER of cells incubated with EPEC did not differsignificantly from the control Pre- and coincubation withE faecium reversed the decreased TER of ETEC-treatedIPEC-J2 cell monolayers at 4 h and as a trend at 6 h whileTER of EPEC-treated cells was not changed by pre- andcoincubation with E faecium

32 HSP Expression As a stress indicator for the pathogenicchallenge HSP70 expression was tested in both cell lines atthe mRNA and protein levels

321 Caco-2 In the overall analysis the differences betweenthe treatment groups were statistically significant for themRNA expression of HSP70 (119875 = 0001)

After four hours of incubation no differences could beobserved between control cells and cells incubated withEPEC or E faecium (alone or in combination with ETECand EPEC) (Figure 4(a)) However the mRNA expressionof HSP70 was significantly higher in the cells that wereincubated with the ETEC strain alone compared with allother treatmentsThis implied that the coincubation of ETECwith E faecium significantly reduced the increase in mRNAexpression observed during incubation with ETEC alone

These results were reflected by the changes in HSP70protein expression (Figure 4(b))

322 IPEC-J2 For IPEC-J2 cells the mRNA and proteinexpression of HSP70 showed some numerical but no statis-tically significant differences between the treatment groups(Figure 5)

33 IL-8 Expression The cytokine IL-8 was chosen as ameasure of the proinflammatory response of the cells

331 Caco-2 The bacterial incubation significantly affectedthe mRNA expression of IL-8 (119875 = 0008) The mRNAexpression of IL-8 did not differ between the cells incubatedwith E faecium and the control cells However it wassignificantly increased 4 h after incubation with the ETECstrain compared with the control and the E faecium-treatedcells Coincubation with E faecium significantly reducedthis increase (Figure 6(a)) Although a numerical increasewas seen in all groups incubated with bacteria the mRNAexpression did not differ significantly between the othertreatment groups and the control (Figure 6(a))

The changes in mRNA expression were mirrored bysimilar changes in IL-8 protein expression (Figure 6(b))

332 IPEC-J2 The results regarding IL-8 expression by theIPEC-J2 cell line were largely comparable with the resultsobtained for Caco-2 cells In the overall analysis significantdifferences for IL-8 mRNA (119875 = 0001) and protein expres-sion (119875 = 0001) were observed and statistical differences


150

100

50

0

4 6

ControlEnterotoxigenic E coli

Enteropathogenic E coliCoincubation with E faecium

a

a

a

a aa

c

b

b

b b

b

Hours of incubation

TER

(in

of i

nitia

l val

ue at

t=0

h)

Figure 2 TER values of Caco-2 cells in percent of the initial value before the beginning of the experiment (119905 = 0 hours) (means plusmn SEM) Cellmonolayers were treated with various bacterial strains for 4 h and 6 h119873 = 6 independent experiments different letters indicate significantdifferences between the differently treated cells (119875 le 005)

150

200

100

50

0

aa

a aa a

a

aa

a

b bb

Hours of incubation

TER

(in

of i

nitia

l val

ue at

t=0

h)



4 6

Figure 3 TER values of the IPEC-J2 cells in percent of the initial value before the beginning of the experiment (119905 = 0 hours) (means plusmnSEM) Cell monolayers were treated with various bacterial strains for 4 h and 6 h119873 = 6 independent experiments different letters indicatesignificant differences between the differently treated cells (119875 le 005)

between individual groups were as described for Caco-2 cells(Figure 7)

4 Discussion

The aim of this study was to elucidate the effects of theprobiotic E faecium on barrier function and the inflamma-tory response of the intestinal epithelium the latter effectbeing in addition to other functions an important part of theimmune system of the gut To examine whether the probioticstrain could modify the epithelial response to a pathogenicchallenge epithelial cell monolayers were incubated withtwo selected pathogenic E coli strains Our hypothesiswas that epithelial integrity might be improved whereas

the expression of HSP70 and proinflammatory cytokinesmight be reduced because of the action of E faecium

Such ldquochallengerdquo experiments can be carried out invarious ways One approach is to treat the confluent cell mon-olayers directly with the pathogenic bacteria for the wholeduration of the experiment The second is to incubate theepithelial cells with supernatants of the bacterial cultures[23 38]The advantage of using supernatants is the avoidanceof pH decreases in the medium or nutrient competition bythe rapidly growing bacteria [39] However supernatantsor dead bacteria cannot mimic the conditions of livingbacteria and exclude potential direct interactions of livingbacteria with the epithelial cells To include such bacteria-epithelial interactions the experimental design of the present


b b

a

b

b

b

0

1

2

3

4

5

6

7N

orm

aliz

ed fo

ld ex

pres

sion

Control ETEC EPEC

No coincubationCoincubation with E faecium

(a)

0

05

1

15

2

25

3

35

4

45

5

Prot

ein

expr

essio

n (p

gm

L)

b b

a

b

b

b

Control ETEC EPEC


(b)

Figure 4 HSP70 expression in Caco-2 cells after treatment with various bacterial strains (means plusmn SEM) (a) mRNA expression 119873 = 5independent experiments and (b) protein expression 119873 = 4 independent experiments different letters indicate significant differencesbetween the differently treated cells (119875 le 005)

0

05

1

15

2

25

3

Nor

mal

ized

fold

expr

essio

n

Control ETEC EPEC


(a)

0

20

40

60

80

100

120

140

160

180

Prot

ein

expr

essio

n (p

gm

L)

Control ETEC EPEC


(b)

Figure 5 HSP70 expression in IPEC-J2 cells after treatment with various bacterial strains (means plusmn SEM) (a) mRNA expression 119873 = 6independent experiments and (b) protein expression119873 = 4 independent experiments

study included an initial incubation with living bacteria for2 h Thereafter bacteria were killed by gentamicin with theintention of diminishing secondary effects of the bacterialincubation Two intestinal epithelial cell lines were usedporcine IPEC-J2 and human Caco-2 cells

The Caco-2 cell line is well established and has beenused for many years to investigate specific aspects of smallintestinal function including investigations on the effects ofprobiotic bacteria [40 41] Nonetheless it has limitationsbecause of its cancerogenic origin from the colon Theporcine IPEC-J2 cells seem to be a suitable model to mimicin vivo conditions of the small intestine as they have no

tumorous origin and were originally isolated from the smallintestine [30 42] they have been recently used to study otherprobiotic microorganisms [23 39] The second aim of thisstudy has therefore been to compareCaco-2 and IPEC-J2 cellsunder the same experimental conditions to provide furtherevidence for the use of IPEC-J2 cells as a model for smallintestinal simulation

41 TER TER is a compound measure of paracellular andtranscellular resistance and has been used to assess epithelialintegrity in probiotic studies [43 44] In the present studythe TER of both cell lines was slightly increased after 4 h


0

50

100

150

200

250

300N

orm

aliz

ed fo

ld ex

pres

sion

b b

a

bb

b

Control ETEC EPEC


(a)

Prot

ein

expr

essio

n (p

gm

L)

0

10

20

30

40

50

60

b b

a

bb

b

Control ETEC EPEC


(b)

Figure 6 IL-8 expression in Caco-2 cells after treatment with various bacterial strains (means plusmn SEM) (a) mRNA expression 119873 = 5independent experiments and (b) protein expression 119873 = 5 independent experiments different letters indicate significant differencesbetween the differently treated cells (119875 le 005)

0

2

4

6

8

10

12

14

Nor

mal

ized

fold

expr

essio

n

b b

a

b

bb

Control ETEC EPEC


(a)

0

10

20

30

40

50

60

70

80

90

Prot

ein

expr

essio

n (p

gm

L)

b b

a

bb b

Control ETEC EPEC


(b)

Figure 7 IL-8 expression in IPEC-J2 cells after treatment with various bacterial strains (means plusmn SEM) (a) mRNA expression 119873 = 6independent experiments and (b) protein expression 119873 = 6 independent experiments different letters indicate significant differencesbetween the differently treated cells (119875 le 005)

of incubation with E faecium alone but significantly onlyin Caco-2 cells These results are in agreement with dataof earlier studies showing that various probiotic bacterialstrains had either no or an enhancing effect on the TER ofintestinal epithelial cells of various origins [45ndash47] In porcineIPEC-J2 cells no enhancing effect has been reported so far[23] The ETEC strain but not the EPEC strain significantlyreduced TER in both cell lines compared with the controlthis indicates a change in the epithelial integrity as shownpreviously by other authors [48 49] This decrease was

inhibited when cell monolayers were (pre- and) coincubatedwith the E faecium strain Similar results have been obtainedfor other probiotic strains in in vitro infection studies withvarious intestinal cell lines of human origin such as T84HT29 or Caco-2 cells [43 44 50] In a porcine cell model(IPEC-1) Lactobacillus sobrius DSM 16698 also inhibited thedecrease of TER caused by an ETEC strain [51]

In former studies a probiotic action to prevent the TERdecrease induced by ETEC has been correlated with theinhibition of E coli adhesion which is the first step of


ETEC infection [52] Another possibility by which E faeciumcould prevent the decrease of TER after ETEC infectionmight be related to tight junction (TJ) protein expression andlocalization or to the cytoskeletal organization [51]

These parameters have not been assessed here but will bethe target of further studies

42 HSP70 HSPs protect cells tissues and organs againstvarious types of stress factors by reducing or avoiding thestress-induced denaturation of proteins Gene expression ofinducible HSP is triggered by many different stressors andis mainly regulated at the transcriptional level [53ndash55] Inthe gastrointestinal tract the expression of inducible HSP ismarkedly influenced by the intestinal microbiota [56 57]

The inducible form of HSP70 which was assessed in thepresent study is often considered to be cytoprotective asits induction by minor stressors can protect the tissue frommajor challenges [58 59] Some probiotic bacteria obviouslyactivate this cytoprotective mechanism as part of their modeof action [23 60 61] E faecium alone however did notalter expression of HSP70 in the present study indicatingthat different probiotics rely on different mechanisms andpathways to exert their effects on the host [15 60]

However in the Caco-2 cells the expression of HSP70was significantly increased in the cells incubated with theETECThis increase could be prevented by probiotic pre- andcoincubation In the IPEC-J2 cells a similar tendency couldbe observed

Although as stated above the upregulation of HSP70 isoften interpreted as a positive effect because of its cytoprotec-tive effects evidence has also been presented suggesting thatHSP70 expression is an indicator of pathological stress [27]

In conjunctionwith the effects onTER and the proinflam-matory cytokine response the increased expression ofHSP70after pathogenic challenge indicates a stress reaction of theintestinal cells As such the reduced increase in HSP70 afterpre- and coincubation with E faecium in the present studysuggests that cells are less damaged by ETEC

43 IL-8 The proinflammatory cytokine response in thegastrointestinal tract is influenced by the gut microbiotaAmong others the cytokine expression of epithelial cellscan be modulated by probiotic strains [21 40 62] Alteredcytokine release in turn can regulate the structure andfunction of TJ and cytoskeleton [63 64] and the transportproperties of epithelial cells [65 66]

In the present study IL-8 has been chosen as a representa-tive cytokine of the epithelial proinflammatory response IL-8 belongs to the proinflammatory ldquochemokinerdquo family and isreported to induce neutrophil and T-lymphocyte chemotaxisneutrophil activation and the enhanced expression of neu-trophil adhesion molecules [67 68] One of the responses ofintestinal epithelial cells as part of the innate immune systemto various inflammatory stimuli is the production of IL-8[69]

In the present study IL-8 expression was considerablyincreased when the cells were incubated with ETEC this

increase was abrogated by concomitant incubation with Efaecium Similar reductions of proinflammatory responsesto a pathogen by probiotic strains have been observed inother in vivo and in vitro models [23 41 51 70] In somecases this was accompanied by the maintenance of epithelialbarrier function [70] In porcine intestinal epithelial cellsvarious lactobacilli and bacilli strains counteract the increasein IL-8 and other proinflammatory cytokines elicited bystimulation with ETEC S typhimurium oxidative stress orlipopolysaccharide In some cases this is associated with theprotection of the epithelial barrier [23 39 71]

Although IL-8 secretion is part of the innate immuneresponse aimed at the elimination of pathogens the persistentproduction of IL-8 accompanied by the constant infiltra-tion of neutrophils leads to massive epithelial cell damage[72] which is one cause of diarrhea Several studies havedemonstrated that epithelial damage can be prevented byinterventions that suppress the IL-8 levels in IBD [73 74]This could also be an approach for reducing epithelial damageand diarrhea in acute infections such as ETEC and herewe consider it to be one of the positive effects of healthymicrobiota [61] Taking this into account the effects of Efaecium observed in the present study indicate a protectiveeffect of this probiotic in acute intestinal inflammationinduced by ETEC

The exact mechanisms by which E faecium exerts itsinfluence on cytokine secretion have to be further investi-gated

5 Conclusion

Preincubation with the probiotic E faecium abrogates orreduces all examined effects induced by the ETEC such asthe HSP70 stress response the elevated expression of theproinflammatory cytokine IL-8 and the decrease in TER asa measure of epithelial integrity

A key feature of the intestinal immune system is its abilityto protect against pathogens while avoiding a destructiveinflammatory response An exaggerated proinflammatorycytokine secretion such as IL-8 leads to disease states and inthis case a reduction of proinflammatory cytokines togetherwith the reduction of HSP70 expression and the preventionof potential epithelial damage might alleviate symptomsindicating a positive effect by the probiotic The underlyingmechanisms will be the subject of further studies

Both cell lines react in a similar manner to incubationwith pathogens and the probiotic Therefore the IPEC-J2cell line can be considered as a reliable model for studyingthe effects of probiotics on the protection of the intestinalepithelium from stressful conditions and inflammation Fur-thermore the parallel response in the two cell lines underlinesthe general transferability of the effects of E faecium seen inthe present study

Conflict of Interests

The authors declare that there is no conflict of interestsregarding the publication of this paper


Acknowledgments

The authors thank Martin Grunau Katharina Wolf andUrsula Scholz for technical assistance with the cell culturestudies and themRNA and protein analyses and express theirgratitude to the members of the SFB 852 This work wassupported by the Deutsche Forschungsgemeinschaft GrantSFB 8521 and by the Sonnenfeld-Stiftung

References

[1] T Arvola K Laiho S Torkkeli et al ldquoProphylactic Lactobacil-lus GG reduces antibiotic-associated diarrhea in children withrespiratory infections a randomized studyrdquo Pediatrics vol 104no 5 article e64 1999

[2] S Guandalini L Pensabene M A Zikri et al ldquoLactobacillusGG administered in oral rehydration solution to childrenwith acute diarrhea a multicenter European trialrdquo Journal ofPediatric Gastroenterology and Nutrition vol 30 no 1 pp 54ndash60 2000

[3] C M Surawicz L V McFarland R N Greenberg et alldquoThe search for a better treatment for recurrent Clostridiumdifficile disease use of high-dose vancomycin combined withSaccharomyces boulardiirdquoClinical Infectious Diseases vol 31 no4 pp 1012ndash1017 2000

[4] H Szajewska M Kotowska J Z Mrukowicz M ArmanskaandWMikolajczyk ldquoEfficacy of LactobacillusGG in preventionof nosocomial diarrhea in infantsrdquoThe Journal of Pediatrics vol138 no 3 pp 361ndash365 2001

[5] S R Konstantinov H Smidt A D L Akkermans et alldquoFeeding of Lactobacillus sobrius reduces Escherichia coli F4levels in the gut and promotes growth of infected pigletsrdquo FEMSMicrobiology Ecology vol 66 no 3 pp 599ndash607 2008

[6] C Agostoni I Axelsson C Braegger et al ldquoProbiotic bac-teria in dietetic products for infants a commentary by theESPGHAN Committee on Nutritionrdquo The Journal of pediatricgastroenterology and nutrition vol 38 no 4 pp 365ndash374 2004

[7] C Alexopoulos A Karagiannidis S K Kritas C Boscos IE Georgoulakis and S C Kyriakis ldquoField evaluation of abioregulator containing live Bacillus cereus spores on healthstatus and performance of sows and their littersrdquo Journal ofVeterinary Medicine Series A Physiology Pathology ClinicalMedicine vol 48 no 3 pp 137ndash145 2001

[8] G Breves C Walter M Burmester and B Schroder ldquoIn vitrostudies on the effects of Saccharomyces boulardii and Bacilluscereus var toyoi on nutrient transport in pig jejunumrdquo Journalof Animal Physiology and Animal Nutrition vol 84 no 1-2 pp9ndash20 2000

[9] M Kirchgessner F X Roth U Eidelsburger and B GedekldquoThe nutritive efficiency of Bacillus cereus as a probiotic inthe raising of piglets 1 Effect on the growth parameters andgastrointestinal environmentrdquo Archiv fur Tierernahrung vol44 no 2 pp 111ndash121 1993

[10] D Taras W Vahjen M Macha and O Simon ldquoPerformancediarrhea incidence and occurrence of Escherichia coli viru-lence genes during long-term administration of a probioticEnterococcus faecium strain to sows and pigletsrdquo Journal ofAnimal Science vol 84 no 3 pp 608ndash617 2006

[11] A Zeyner and E Boldt ldquoEffects of a probiotic Enterococcusfaecium strain supplemented from birth to weaning on diar-rhoea patterns and performance of pigletsrdquo Journal of Animal

Physiology and Animal Nutrition vol 90 no 1-2 pp 25ndash312006

[12] P Buydens and S Debeuckelaere ldquoEfficacy of SF 68 in thetreatment of acute diarrhea A placebo-controlled trialrdquo Scan-dinavian Journal of Gastroenterology vol 31 no 9 pp 887ndash8911996

[13] P F Wunderlich L Braun L Fumagalli et al ldquoDouble-blindreport on the efficacy of lactic acid-producing enterococcusSF68 in the prevention of antibiotic-associated diarrhoea andin the treatment of acute diarrhoeardquo Journal of InternationalMedical Research vol 17 no 4 pp 333ndash338 1989

[14] A Di Mauro J Neu G Riezzo et al ldquoGastrointestinal functiondevelopment and microbiotardquo Italian Journal of Pediatrics vol39 no 1 article 15 2013

[15] P M Sherman J C Ossa and K Johnson-Henry ldquoUnravelingmechanisms of action of probioticsrdquo Nutrition in ClinicalPractice vol 24 no 1 pp 10ndash14 2009

[16] E Isolauri M Kaila T Arvola et al ldquoDiet during rotavirusenteritis affects jejunal permeability to macromolecules insuckling ratsrdquo Pediatric Research vol 33 no 6 pp 548ndash5531993

[17] L Khailova S K Mount Patrick K M Arganbright MD Halpern T Kinouchi and B Dvorak ldquoBifidobacteriumbifidum reduces apoptosis in the intestinal epithelium innecrotizing enterocolitisrdquoTheAmerican Journal of PhysiologymdashGastrointestinal and Liver Physiology vol 299 no 5 pp G1118ndashG1127 2010

[18] PMangell P NejdforsMWang et al ldquoLactobacillus plantarum299v inhibits Escherichia coli-induced intestinal permeabilityrdquoDigestive Diseases and Sciences vol 47 no 3 pp 511ndash516 2002

[19] K C Johnson-Henry K A Donato G Shen-Tu M Gordan-pour and P M Sherman ldquoLactobacillus rhamnosus strain GGprevents enterohemorrhagic Escherichia coli O157H7-inducedchanges in epithelial barrier functionrdquo Infection and Immunityvol 76 no 4 pp 1340ndash1348 2008

[20] P M Sherman K C Johnson-Henry H P Yeung P S CNgo J Goulet and T A Tompkins ldquoProbiotics reduce entero-hemorrhagic Escherichia coli O157H7- and enteropathogenicE coli O127H6-induced changes in polarized T84 epithelialcell monolayers by reducing bacterial adhesion and cytoskeletalrearrangementsrdquo Infection and Immunity vol 73 no 8 pp5183ndash5188 2005

[21] B Bahrami S Macfarlane and G T Macfarlane ldquoInductionof cytokine formation by human intestinal bacteria in gutepithelial cell linesrdquo Journal of AppliedMicrobiology vol 110 no1 pp 353ndash363 2011

[22] G Perdigon S Alvarez M Rachid G Aguero andN GobbatoldquoImmune system stimulation by probioticsrdquo Journal of DairyScience vol 78 no 7 pp 1597ndash1606 1995

[23] E Paszti-Gere K Szeker E Csibrik-Nemeth et al ldquoMetabo-lites of Lactobacillus plantarum 2142 prevent oxidative stress-induced overexpression of proinflammatory cytokines in IPEC-J2 cell linerdquo Inflammation vol 35 no 4 pp 1487ndash1499 2012

[24] D L Arvans S R Vavricka H Ren et al ldquoLuminal bacterialflora determines physiological expression of intestinal epithelialcytoprotective heat shock proteins 25 and 72rdquo The AmericanJournal of PhysiologymdashGastrointestinal and Liver Physiologyvol 288 no 4 pp G696ndashG704 2005

[25] S R Choi S A Lee Y J Kim C Y Ok H J Lee and K BHahm ldquoRole of heat shock proteins in gastric inflammationand ulcer healingrdquo Journal of physiology and pharmacology


an official journal of the Polish Physiological Society vol 60 pp5ndash17 2009

[26] M J Ropeleski J Tang M M Walsh-Reitz M W Muschand E B Chang ldquoInterleukin-11-induced heat shock protein25 confers intestinal epithelial-specific cytoprotection fromoxidant stressrdquo Gastroenterology vol 124 no 5 pp 1358ndash13682003

[27] K Sepponen and A R Poso ldquoThe inducible form of heatshock protein 70 in the serum colon and small intestine of thepig comparison to conventional stress markersrdquoTheVeterinaryJournal vol 171 no 3 pp 519ndash524 2006

[28] A Youakim and M Ahdieh ldquoInterferon-120574 decreases barrierfunction inT84 cells by reducingZO-1 levels anddisrupting api-cal actinrdquoThe American Journal of PhysiologymdashGastrointestinaland Liver Physiology vol 276 no 5 pp G1279ndashG1288 1999

[29] S Klingspor H Martens D Caushi S Twardziok J RAschenbach and U Lodemann ldquoCharacterization of the effectsof Enterococcus faecium on intestinal epithelial transport prop-erties in pigletsrdquo Journal of Animal Science vol 91 no 4 pp1707ndash1718 2013

[30] P SchierackMNordhoffM Pollmann et al ldquoCharacterizationof a porcine intestinal epithelial cell line for in vitro studiesof microbial pathogenesis in swinerdquo Histochemistry and CellBiology vol 125 no 3 pp 293ndash305 2006

[31] C Gabler E Ephraim C Holder R Einspanier and MSchmidt ldquoHigher mRNA expression of proinflammatory fac-tors in immune cells of piglets in probiotic group afterSalmonella infectionrdquo inBook of Abstracts of the satellitemeetingof the 2nd European Congress of Immunology Berlin 2009 p 402009

[32] U Lodemann R Einspanier F Scharfen H Martens and ABondzio ldquoEffects of zinc on epithelial barrier properties andviability in a human and a porcine intestinal cell culturemodelrdquoToxicology in Vitro vol 27 no 2 pp 834ndash843 2013

[33] J M Rhoads W Chen P Chu H M Berschneider R AArgenzio and A M Paradiso ldquoL-Glutamine and L-asparaginestimulate Na+-H+ exchange in porcine jejunal enterocytesrdquoThe American Journal of PhysiologymdashGastrointestinal and LiverPhysiology vol 266 no 5 part 1 pp G828ndashG838 1994

[34] S HuM J Ciancio M Lahav et al ldquoTranslational inhibition ofcolonic epithelial heat shock proteins by IFN-gamma and TNF-alpha in intestinal inflammationrdquoGastroenterology vol 133 no6 pp 1893ndash1904 2007

[35] A Parlesak D Haller S Brinz A Baeuerlein and C BodeldquoModulation of cytokine release by differentiated CACO-2 cellsin a compartmentalized coculture model with mononuclearleucocytes and nonpathogenic bacteriardquo Scandinavian Journalof Immunology vol 60 no 5 pp 477ndash485 2004

[36] C Bernardini A Zannoni M E Turba et al ldquoEffects of 50 Hzsinusoidal magnetic fields on Hsp27 Hsp70 Hsp90 expressionin porcine aortic endothelial cells (PAEC)rdquo Bioelectromagneticsvol 28 no 3 pp 231ndash237 2007

[37] T G Ramsay and T J Caperna ldquoOntogeny of adipokine expres-sion in neonatal pig adipose tissuerdquo Comparative Biochemistryand PhysiologymdashPart AMolecular amp Integrative Physiology vol152 no 1 pp 72ndash78 2009

[38] E Paszti-Gere E Csibrik-Nemeth K Szeker et al ldquoLacto-bacillus plantarum 2142 prevents intestinal oxidative stress inoptimized in vitro systemsrdquo Acta Physiologica Hungarica vol100 no 1 pp 89ndash98 2013

[39] C C Aperce T E Burkey B KuKanich B A Crozier-DodsonS S Dritz and J E Minton ldquoInteraction of Bacillus species

and Salmonella enterica serovar Typhimurium in immune orinflammatory signaling from swine intestinal epithelial cellsrdquoJournal of Animal Science vol 88 no 5 pp 1649ndash1656 2010

[40] C M Carey andM Kostrzynska ldquoLactic acid bacteria and bifi-dobacteria attenuate the proinflammatory response in intesti-nal epithelial cells induced by salmonella enterica serovarTyphimuriumrdquo Canadian Journal of Microbiology vol 59 no1 pp 9ndash17 2013

[41] K Madsen A Cornish P Soper et al ldquoProbiotic bacteriaenhance murine and human intestinal epithelial barrier func-tionrdquo Gastroenterology vol 121 no 3 pp 580ndash591 2001

[42] A J Brosnahan and D R Brown ldquoPorcine IPEC-J2 intestinalepithelial cells in microbiological investigationsrdquo VeterinaryMicrobiology vol 156 no 3-4 pp 229ndash237 2012

[43] J-M Otte and D K Podolsky ldquoFunctional modulation ofenterocytes by gram-positive and gram-negative microorgan-ismsrdquoAmerican Journal of Physiology Gastrointestinal and LiverPhysiology vol 286 no 4 pp G613ndashG626 2004

[44] A A Zyrek C Cichon S Helms C Enders U Sonnenbornand M A Schmidt ldquoMolecular mechanisms underlying theprobiotic effects of Escherichia coliNissle 1917 involve ZO-2 andPKCzeta redistribution resulting in tight junction and epithelialbarrier repairrdquo Cellular Microbiology vol 9 no 3 pp 804ndash8162007

[45] J B Ewaschuk H Diaz L Meddings et al ldquoSecreted bioactivefactors fromBifidobacterium infantis enhance epithelial cell bar-rier functionrdquoAmerican Journal of PhysiologymdashGastrointestinaland Liver Physiology vol 295 no 5 pp G1025ndashG1034 2008

[46] S Resta-Lenert and K E Barrett ldquoLive probiotics protectintestinal epithelial cells from the effects of infection withenteroinvasive Escherichia coli (EIEC)rdquo Gut vol 52 no 7 pp988ndash997 2003

[47] P Lopez I Gonzalez-Rodrıguez B Sanchez et al ldquoInteractionof Bifidobacterium bifidum LMG13195 with HT29 Cells Influ-ences regulatory-T-cell-associated chemokine receptor expres-sionrdquo Applied and Environmental Microbiology vol 78 no 8pp 2850ndash2857 2012

[48] 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 11 pages 2010

[49] M Roselli M S Britti I Le Huerou-Luron H Marfaing WY Zhu and E Mengheri ldquoEffect of different plant extracts andnatural substances (PENS) against membrane damage inducedby enterotoxigenic Escherichia coli K88 in pig intestinal cellsrdquoToxicology in Vitro vol 21 no 2 pp 224ndash229 2007

[50] J S Ko H R Yang J Y Chang and J K Seo ldquoLacto-bacillus plantarum inhibits epithelial barrier dysfunction andinterlukin-8 secretion induced by tumor necrosis factor-120572rdquoWorld Journal of Gastroenterology vol 13 no 13 pp 1962ndash19652007

[51] M Roselli A Finamore M S Britti et al ldquoThe novelporcine Lactobacillus sobrius strain protects intestinal cellsfrom enterotoxigenic Escherichia coli K88 infection and pre-vents membrane barrier damagerdquo Journal of Nutrition vol 137no 12 pp 2709ndash2716 2007

[52] G Gonzalez-Ortiz R G Hermes R Jimenez-Dıaz J F Perezand S M Martın-Orue ldquoScreening of extracts from naturalfeed ingredients for their ability to reduce enterotoxigenicEscherichia coli (ETEC) K88 adhesion to porcine intestinal


epithelial cell-line IPEC-J2rdquo Veterinary Microbiology vol 167no 3-4 pp 494ndash499 2013

[53] S Lindquist and E A Craig ldquoThe heat-shock proteinsrdquoAnnualReview of Genetics vol 22 pp 631ndash677 1988

[54] H M Beere ldquoDeath versus survival functional interactionbetween the apoptotic and stress-inducible heat shock proteinpathwaysrdquo The Journal of Clinical Investigation vol 115 no 10pp 2633ndash2639 2005

[55] V L Gabai and M Y Sherman ldquoInvited review Interplaybetween molecular chaperones and signaling pathways in sur-vival of heat shockrdquo Journal of Applied Physiology vol 92 no 4pp 1743ndash1748 2002

[56] S Hu Y Wang L Lichtenstein et al ldquoRegional differencesin colonic mucosa-associated microbiota determine the phys-iological expression of host heat shock proteinsrdquo AmericanJournal of PhysiologymdashGastrointestinal and Liver Physiologyvol 299 no 6 pp G1266ndashG1275 2010

[57] K Dokladny P L Moseley and T Y Ma ldquoPhysiologicallyrelevant increase in temperature causes an increase in intestinalepithelial tight junction permeabilityrdquo American Journal ofPhysiologymdashGastrointestinal and Liver Physiology vol 290 no2 pp G204ndashG212 2006

[58] D L Arvans S R Vavricka H Ren et al ldquoLuminal bacterialflora determines physiological expression of intestinal epithelialcytoprotective heat shock proteins 25 and 72rdquoAmerican Journalof PhysiologymdashGastrointestinal and Liver Physiology vol 288no 4 pp G696ndashG704 2005

[59] J J Malago J F Koninkx and J E van Dijk ldquoThe heat shockresponse and cytoprotection of the intestinal epitheliumrdquo CellStress and Chaperone vol 7 no 2 pp 191ndash199 2002

[60] J F J G Koninkx and J J Malago ldquoThe protective potency ofprobiotic bacteria and their microbial products against entericinfections-reviewrdquo Folia Microbiologica vol 53 no 3 pp 189ndash194 2008

[61] J J Malago P C Tooten and J F Koninkx ldquoAnti-inflammatoryproperties of probiotic bacteria on Salmonella-induced IL-8synthesis in enterocyte-like Caco-2 cellsrdquo Beneficial microbesvol 1 no 2 pp 121ndash130 2010

[62] B Siepert N Reinhardt S Kreuzer et al ldquoEnterococcus fae-ciumNCIMB 10415 supplementation affects intestinal immune-associated gene expression in post-weaning pigletsrdquo VeterinaryImmunology and Immunopathology vol 157 no 1-2 pp 65ndash772014

[63] M Bruewer A Luegering T Kucharzik et al ldquoProinflamma-tory cytokines disrupt epithelial barrier function by apoptosis-independent mechanismsrdquoThe Journal of Immunology vol 171no 11 pp 6164ndash6172 2003

[64] A Nusrat J R Turner and J L Madara ldquoMolecular physiologyand pathophysiology of tight junctions IV Regulation oftight junctions by extracellular stimuli nutrients cytokinesand immune cellsrdquo The American Journal of PhysiologymdashGastrointestinal and Liver Physiology vol 279 no 5 pp G851ndashG857 2000

[65] D C Chiossone P L Simon and P L Smith ldquoInterleukin-1effects on rabbit ileal mucosal ion transport in vitrordquo EuropeanJournal of Pharmacology vol 180 no 2-3 pp 217ndash228 1990

[66] J Hardin K Kroeker B Chung and D G Gall ldquoEffect ofproinflammatory interleukins on jejunal nutrient transportrdquoGut vol 47 no 2 pp 184ndash191 2000

[67] B Sherry andA Cerami ldquoSmall cytokine super-familyrdquoCurrentOpinion in Immunology vol 3 no 1 pp 56ndash60 1991

[68] S E Crowe L Alvarez M Dytoc et al ldquoExpression ofinterleukin 8 and CD54 by human gastric epithelium afterHelicobacter pylori infection in vitrordquo Gastroenterology vol108 no 1 pp 65ndash74 1995

[69] D I Sonnier S R Bailey R M Schuster A B Lentsch and TA Pritts ldquoTNF-120572 induces vectorial secretion of IL-8 in Caco-2 cellsrdquo Journal of Gastrointestinal Surgery vol 14 no 10 pp1592ndash1599 2010

[70] J Ewaschuk R Endersby D Thiel et al ldquoProbiotic bacteriaprevent hepatic damage and maintain colonic barrier functionin a mouse model of sepsisrdquo Hepatology vol 46 no 3 pp 841ndash850 2007

[71] K A Skjolaas T E Burkey S S Dritz and J E MintonldquoEffects of Salmonella enterica serovar Typhimurium or serovarCholeraesuis Lactobacillus reuteri and Bacillus licheniformison chemokine and cytokine expression in the swine jeju-nal epithelial cell line IPEC-J2rdquo Veterinary Immunology andImmunopathology vol 115 no 3-4 pp 299ndash308 2007

[72] D-M McCafferty and I J Zeitlin ldquoShort chain fatty acid-induced colitis in micerdquo International Journal of Tissue Reac-tions vol 11 no 4 pp 165ndash168 1989

[73] J M Harig K H Soergel R A Komorowski and C MWood ldquoTreatment of diversion colitis with short-chain-fattyacid irrigationrdquoThe New England Journal of Medicine vol 320no 1 pp 23ndash28 1989

[74] W Scheppach H Sommer T Kirchner et al ldquoEffect of butyrateenemas on the colonic mucosa in distal ulcerative colitisrdquoGastroenterology vol 103 no 1 pp 51ndash56 1992

Submit your manuscripts athttpwwwhindawicom

Stem CellsInternational

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014


MEDIATORSINFLAMMATION

of


Behavioural Neurology

EndocrinologyInternational Journal of



Disease Markers


BioMed Research International

OncologyJournal of



Oxidative Medicine and Cellular Longevity


PPAR Research

The Scientific World JournalHindawi Publishing Corporation httpwwwhindawicom Volume 2014

Immunology ResearchHindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Journal of

ObesityJournal of



Computational and Mathematical Methods in Medicine

OphthalmologyJournal of


Diabetes ResearchJournal of



Research and TreatmentAIDS


Gastroenterology Research and Practice


Parkinsonrsquos Disease

Evidence-Based Complementary and Alternative Medicine

Volume 2014Hindawi Publishing Corporationhttpwwwhindawicom


and epithelial and immune cells of the intestinal wall isextremely complex [14 15] and many of the underlyingsignaling mechanisms are still only partially understood

In vivomodels of gastrointestinal infections have demon-strated a positive effect of various probiotic feed additives onthe functional barrier of the intestine [16ndash18] These resultsare supported by data from in vitro cell culture infectionmodels in which probiotic strains prevent or amelioratedamage to epithelial integrity by a pathogenic challenge [1920] Furthermore the immunological response of themucosacan be influenced by probiotic strains which modulate therelease of cytokines amongst other effects [21 22] Forexample culture supernatant of Lactobacillus plantarum 2142had a suppressive effect on the interleukin-8 (IL-8) and tumornecrosis factor-120572 (TNF-120572) levels induced by oxidative stressin IPEC-J2 cells [23]

The expression of heat shock proteins (HSPs) a set ofproteins that are involved in many regulatory pathways andthat serve as chaperones for preserving cellular proteins canbe influenced by the intestinal microbiota and by probiotics[24ndash26] Some of these HSPs such as the inducible formof HSP70 are upregulated by several noxious conditionsand can therefore be considered as cellular stress indicators[27] HSPs and cytokines in turn are able to regulatebarrier properties by influencing tight junction proteins andthe structure and function of the cytoskeleton or transportproperties of the epithelium [24 28]

In previous studies feed supplementation with E fae-cium has been demonstrated to increase the absorptive andsecretory capacity and improve barrier function of the smallintestinal epithelium of piglets [29 30] Furthermore theproinflammatory cytokine IL-1120572 which is expressed in higheramounts in Peyerrsquos patches of E faecium-treated piglets [31]increases chloride secretion in the small intestine of pigs [29]

The hypothesis of the current study is that intestinal epi-thelial cells play an important role in innate immuneresponses during enteric infections and that E faecium inturn modulates the severity of enteric infections via thealtered generation of proinflammatory cytokines and HSPsin intestinal cells Thus the aim of the present study has beento investigate the influence of the probiotic E faecium andtwo different pathogenic E coli strains on the HSP and proin-flammatory cytokine responses and the epithelial integrityof two intestinal epithelial cell lines We have further testedwhether pre- and coincubation with E faecium change theepithelial cell response to pathogenic E coli strains

2 Materials and Methods

21 Cells and Culture Conditions The cell cultivation isdescribed in detail in Lodemann et al [32] The humanepithelial intestinal cell line from colorectal adenocarcinomaCaco-2 (ATCC Catalog number HTB-37 ATCC Manas-sas USA passages 37ndash45) was used as a model for thehuman small intestine The porcine intestinal epithelial cellline (IPEC-J2 passages 73ndash79) was used as a model forthe pig small intestine This cell line was established fromthe jejunum of a newborn pig [33] and kindly provided

by Professor Dr Anthony Blikslager (North Carolina StateUniversity USA) The cells routinely tested negative formycoplasma contamination

Cells for the experiments were allowed to differentiate for14 days (IPEC-J2) or 21 days (Caco-2) On the day prior toexperiments the cells were fed with serum- and antibiotic-free media

22 Bacterial Strains Three different bacterial strains wereused for the experiments (1) the probiotic strain Enterococ-cus faecium NCIMB 10415 (cultivated from Cylactin DSMHeerlen the Netherlands) (2) the enterotoxigenic E coliIMT4818 (ETEC isolated from a two-week-old piglet withenteritis O149K91K88 (F4) and found to be positive for thepresence of virulence genes est-1a est-2 (genes coding forheat stable enterotoxins I and II) and elt-1ab (gene codingfor heat labile enterotoxin I) by the polymerase chain reaction(PCR)) and (3) the human enteropathogenicE coliE234869(EPEC serotypeO127H6 positive for the eae gene coding forthe E coli attaching-effacing factor)

The E faecium NCIMB 10415 strain was cultivated inbrain-heart infusion (BHI) broth (OXOID GmbH WeselGermany) and the E coli strains in LB medium accordingto Miller containing 10 gL tryptone (OXOID GmbH WeselGermany) 5 gL yeast extract (OXOID GmbH Wesel Ger-many) and 10 gL NaCl at a pH of 70

After overnight incubation of the cells at 37∘C sub-cultures of bacteria were grown for 3 to 4 h until mid-log phase and then centrifuged Cell pellets were washedtwice in phosphate-buffered saline (PBS Biochrom BerlinGermany) The bacteria were resuspended in antibiotic- andserum-free Caco-2 or IPEC-J2 cell culture medium to reacha concentration of asymp108 colony-forming units (CFU)mL

The optical density was measured to determine the con-centration of bacterial cellsThemeasurement was confirmedby serial dilution on agar plates The intestinal cells wereinfected with 106 bacteria per cell culture insert (112 cm2)or per well (191 cm2) corresponding to a multiplicity ofinfection (MOI) of about 10 bacteria per seeded cell Thebacteria were added to the apical pole of the cells

23 Experimental Setup and Procedure For each experimentthe cell monolayers for the real-time quantitative PCRs (RT-qPCR) for enzyme-linked immunosorbent assay (ELISA)and for the transepithelial electrical resistance (TER) mea-surements were incubated for 2 hwith the respective bacterialstrains (ETEC EPEC or E faecium) at a concentration of106 bacteria per cell culture insert or well (see Figure 1) Thecontrol cells received the equivalent amount of bacteria-freemedium Two hours after the bacterial incubation the cellswere washed twice with gentamicin-supplemented media towash out and kill the bacteria and gentamicin-supplementedmedia were added Gentamicin was added to the media ata concentration of 50120583gmL (Biochrom Berlin Germany)The cells were incubated for various periods of time withregard to the different parameters measured the exact timeperiods being given in Figure 1 under the specific headingsWhen the cells were incubated with the probiotic and either




for the control


TER measurement

Sampling for ELISA

























3 Results














150

100

50

0

4 6



a

a

a

a aa

c

b

b

b b

b

Hours of incubation

TER

(in

of i

nitia

l val

ue at

t=0

h)


150

200

100

50

0

aa

a aa a

a

aa

a

b bb

Hours of incubation

TER

(in

of i

nitia

l val

ue at

t=0

h)



4 6



4 Discussion





b b

a

b

b

b

0

1

2

3

4

5

6

7N

orm

aliz

ed fo

ld ex

pres

sion

Control ETEC EPEC


(a)

0

05

1

15

2

25

3

35

4

45

5

Prot

ein

expr

essio

n (p

gm

L)

b b

a

b

b

b

Control ETEC EPEC


(b)


0

05

1

15

2

25

3

Nor

mal

ized

fold

expr

essio

n

Control ETEC EPEC


(a)

0

20

40

60

80

100

120

140

160

180

Prot

ein

expr

essio

n (p

gm

L)

Control ETEC EPEC


(b)







0

50

100

150

200

250

300N

orm

aliz

ed fo

ld ex

pres

sion

b b

a

bb

b

Control ETEC EPEC


(a)

Prot

ein

expr

essio

n (p

gm

L)

0

10

20

30

40

50

60

b b

a

bb

b

Control ETEC EPEC


(b)


0

2

4

6

8

10

12

14

Nor

mal

ized

fold

expr

essio

n

b b

a

b

bb

Control ETEC EPEC


(a)

0

10

20

30

40

50

60

70

80

90

Prot

ein

expr

essio

n (p

gm

L)

b b

a

bb b

Control ETEC EPEC


(b)



















5 Conclusion







Acknowledgments


References






















































































of






Disease Markers



OncologyJournal of





PPAR Research



Journal of

ObesityJournal of



















for the control


TER measurement

Sampling for ELISA

























3 Results














150

100

50

0

4 6



a

a

a

a aa

c

b

b

b b

b

Hours of incubation

TER

(in

of i

nitia

l val

ue at

t=0

h)


150

200

100

50

0

aa

a aa a

a

aa

a

b bb

Hours of incubation

TER

(in

of i

nitia

l val

ue at

t=0

h)



4 6



4 Discussion





b b

a

b

b

b

0

1

2

3

4

5

6

7N

orm

aliz

ed fo

ld ex

pres

sion

Control ETEC EPEC


(a)

0

05

1

15

2

25

3

35

4

45

5

Prot

ein

expr

essio

n (p

gm

L)

b b

a

b

b

b

Control ETEC EPEC


(b)


0

05

1

15

2

25

3

Nor

mal

ized

fold

expr

essio

n

Control ETEC EPEC


(a)

0

20

40

60

80

100

120

140

160

180

Prot

ein

expr

essio

n (p

gm

L)

Control ETEC EPEC


(b)







0

50

100

150

200

250

300N

orm

aliz

ed fo

ld ex

pres

sion

b b

a

bb

b

Control ETEC EPEC


(a)

Prot

ein

expr

essio

n (p

gm

L)

0

10

20

30

40

50

60

b b

a

bb

b

Control ETEC EPEC


(b)


0

2

4

6

8

10

12

14

Nor

mal

ized

fold

expr

essio

n

b b

a

b

bb

Control ETEC EPEC


(a)

0

10

20

30

40

50

60

70

80

90

Prot

ein

expr

essio

n (p

gm

L)

b b

a

bb b

Control ETEC EPEC


(b)



















5 Conclusion







Acknowledgments


References






















































































of






Disease Markers



OncologyJournal of





PPAR Research



Journal of

ObesityJournal of



























3 Results














150

100

50

0

4 6



a

a

a

a aa

c

b

b

b b

b

Hours of incubation

TER

(in

of i

nitia

l val

ue at

t=0

h)


150

200

100

50

0

aa

a aa a

a

aa

a

b bb

Hours of incubation

TER

(in

of i

nitia

l val

ue at

t=0

h)



4 6



4 Discussion





b b

a

b

b

b

0

1

2

3

4

5

6

7N

orm

aliz

ed fo

ld ex

pres

sion

Control ETEC EPEC


(a)

0

05

1

15

2

25

3

35

4

45

5

Prot

ein

expr

essio

n (p

gm

L)

b b

a

b

b

b

Control ETEC EPEC


(b)


0

05

1

15

2

25

3

Nor

mal

ized

fold

expr

essio

n

Control ETEC EPEC


(a)

0

20

40

60

80

100

120

140

160

180

Prot

ein

expr

essio

n (p

gm

L)

Control ETEC EPEC


(b)







0

50

100

150

200

250

300N

orm

aliz

ed fo

ld ex

pres

sion

b b

a

bb

b

Control ETEC EPEC


(a)

Prot

ein

expr

essio

n (p

gm

L)

0

10

20

30

40

50

60

b b

a

bb

b

Control ETEC EPEC


(b)


0

2

4

6

8

10

12

14

Nor

mal

ized

fold

expr

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n

b b

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b

bb

Control ETEC EPEC


(a)

0

10

20

30

40

50

60

70

80

90

Prot

ein

expr

essio

n (p

gm

L)

b b

a

bb b

Control ETEC EPEC


(b)



















5 Conclusion







Acknowledgments


References






















































































of






Disease Markers



OncologyJournal of





PPAR Research



Journal of

ObesityJournal of

















150

100

50

0

4 6



a

a

a

a aa

c

b

b

b b

b

Hours of incubation

TER

(in

of i

nitia

l val

ue at

t=0

h)


150

200

100

50

0

aa

a aa a

a

aa

a

b bb

Hours of incubation

TER

(in

of i

nitia

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ue at

t=0

h)



4 6



4 Discussion





b b

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b

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1

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0

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5

Prot

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(b)


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mal

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expr

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Control ETEC EPEC


(a)

0

20

40

60

80

100

120

140

160

180

Prot

ein

expr

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Control ETEC EPEC


(b)







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50

100

150

200

250

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Control ETEC EPEC


(a)

Prot

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expr

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0

10

20

30

40

50

60

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a

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(b)


0

2

4

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b b

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b

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Control ETEC EPEC


(a)

0

10

20

30

40

50

60

70

80

90

Prot

ein

expr

essio

n (p

gm

L)

b b

a

bb b

Control ETEC EPEC


(b)



















5 Conclusion







Acknowledgments


References






















































































of






Disease Markers



OncologyJournal of





PPAR Research



Journal of

ObesityJournal of

















b b

a

b

b

b

0

1

2

3

4

5

6

7N

orm

aliz

ed fo

ld ex

pres

sion

Control ETEC EPEC


(a)

0

05

1

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3

35

4

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5

Prot

ein

expr

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n (p

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b b

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Control ETEC EPEC


(b)


0

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mal

ized

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Control ETEC EPEC


(a)

0

20

40

60

80

100

120

140

160

180

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ein

expr

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n (p

gm

L)

Control ETEC EPEC


(b)







0

50

100

150

200

250

300N

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aliz

ed fo

ld ex

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sion

b b

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Control ETEC EPEC


(a)

Prot

ein

expr

essio

n (p

gm

L)

0

10

20

30

40

50

60

b b

a

bb

b

Control ETEC EPEC


(b)


0

2

4

6

8

10

12

14

Nor

mal

ized

fold

expr

essio

n

b b

a

b

bb

Control ETEC EPEC


(a)

0

10

20

30

40

50

60

70

80

90

Prot

ein

expr

essio

n (p

gm

L)

b b

a

bb b

Control ETEC EPEC


(b)



















5 Conclusion







Acknowledgments


References






















































































of






Disease Markers



OncologyJournal of





PPAR Research



Journal of

ObesityJournal of

















0

50

100

150

200

250

300N

orm

aliz

ed fo

ld ex

pres

sion

b b

a

bb

b

Control ETEC EPEC


(a)

Prot

ein

expr

essio

n (p

gm

L)

0

10

20

30

40

50

60

b b

a

bb

b

Control ETEC EPEC


(b)


0

2

4

6

8

10

12

14

Nor

mal

ized

fold

expr

essio

n

b b

a

b

bb

Control ETEC EPEC


(a)

0

10

20

30

40

50

60

70

80

90

Prot

ein

expr

essio

n (p

gm

L)

b b

a

bb b

Control ETEC EPEC


(b)



















5 Conclusion







Acknowledgments


References






















































































of






Disease Markers



OncologyJournal of





PPAR Research



Journal of

ObesityJournal of






























5 Conclusion







Acknowledgments


References






















































































of






Disease Markers



OncologyJournal of





PPAR Research



Journal of

ObesityJournal of

















Acknowledgments


References






















































































of






Disease Markers



OncologyJournal of





PPAR Research



Journal of

ObesityJournal of











































































of






Disease Markers



OncologyJournal of





PPAR Research



Journal of

ObesityJournal of
















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