in vitro salmonella lactobacillus strainsdownloads.hindawi.com/journals/grp/2013/973209.pdf ·...

Hindawi Publishing CorporationGastroenterology Research and PracticeVolume 2013 Article ID 973209 6 pageshttpdxdoiorg1011552013973209

Research ArticleIn Vitro Prevention of SalmonellaLipopolysaccharide-Induced Damages in EpithelialBarrier Function by Various Lactobacillus Strains

Chun-Yan Yeung1234 Jen-Shiu Chiang Chiau5 Wai-Tao Chan123 Chun-Bin Jiang12

Mei-Lien Cheng5 Hsuan-Liang Liu3 and Hung-Chang Lee67

1 Division of Gastroenterology and Nutrition Department of Paediatrics Mackay Memorial Hospital Taipei 10449 Taiwan2Mackay Medicine Nursing and Management College Taipei 25245 Taiwan3 Institute of Biotechnology and Department of Chemical Engineering National Taipei University of Technology No 1 Sec 3Chung-Hsiao East Road Taipei 10608 Taiwan

4Department of Medicine Mackay Medical College Taipei 25246 Taiwan5Department of Medical Research Mackay Memorial Hospital Taipei 25160 Taiwan6Division of Gastroenterology and Nutrition Department of Paediatrics Mackay Memorial Hospital No 690 Sec 2Guangfu Road Hsinchu 30071 Taiwan

7Department of Paediatrics Taipei Medical University Taipei 11031 Taiwan

Correspondence should be addressed to Hsuan-Liang Liu f10894ntutedutw and Hung-Chang Lee ped2435ms2mmhorgtw

Received 8 March 2013 Accepted 7 May 2013

Academic Editor Paolo Gionchetti

Copyright copy 2013 Chun-Yan Yeung 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

Background Lactobacillus shows beneficial anti-inflammatory effects on Salmonella infection The maintenance of the tightjunction (TJ) integrity plays an importance role in avoiding bacterial invasion Whether Lactobacillus could be used to regulatethe TJ protein expression and distribution in inflamed intestinal epithelial cells was determined Methods Using the transwellcoculture model Salmonella lipopolysaccharide (LPS) was apically added to polarized Caco-2 cells cocultured with peripheralblood mononuclear cells in the basolateral compartment LPS-stimulated Caco-2 cells were incubated with various Lactobacillusstrains TJ integrity was determined by measuring transepithelial electrical resistance across Caco-2 monolayer Expression andlocalization of TJ proteins (zonula-occludens- (ZO-) 1) were determined by Western blot and immunofluorescence microscopyResults Various strains of Lactobacillus were responsible for the different modulations of cell layer integrity LPS was specificallyable to disrupt epithelial barrier and change the location of ZO-1 Our data demonstrate that Lactobacillus could attenuate thebarrier disruption of intestinal epithelial cells caused by Salmonella LPS administration We showed that Lactobacillus strains areassociated with themaintenance of the tight junction integrity and appearanceConclusion In this study we provide insight that liveprobiotics could improve epithelial barrier properties and this may explain the potential mechanism behind their beneficial effectin vivo

1 Introduction

Salmonella infection is a common cause of human foodpoisoning worldwide and can induce a broad spectrumof diseases from mild diarrhea to typhoid All Salmonellaserotypes share the ability to gain entry to the host throughoral ingestion of contaminated food or water They inducetheir own uptake into intestinal epithelial by Salmonella

pathogenicity island-1 (SPI-1) type 3 secretion system Thisepithelial barrier function can be further weakened by infec-tion with bacteria including S typhimurium effectors [12] Salmonella enterica serovar Typhimurium has developedmeans of breaching the mucosal epithelial barrier by usurp-ing signalingmechanisms within host cells [3] It is likely thatSalmonella induces localized effects on tight junction per-meability during intestinal infections These effects may act

2 Gastroenterology Research and Practice

synergistically with other conditions such as inflammatoryresponses to promote tight junction (TJ) dysfunction [4]At present S typhimurium overcoming the intestinal barrieris the most widely accepted mechanism for their enteringinto host cells especially deeper tissues Thus the mainte-nance of the tight junction integrity of polarized epithelialmonolayer plays an important role in avoiding bacterialinvasion

Besides the disrupted barrier function by S typhimuriumeffectors evidence also showed that lipopolysaccharide (LPS)could induce derangements in intestinal epithelial barrierfunction In our previous study we demonstrated the evi-dence of Salmonella LPS-induced inflammation and epithe-lial barrier dysfunction [5] However the specific TJ respon-sible for the barrier dysfunction has not been identified Byusing polarized epithelial monolayers we might clarify themechanism that Salmonella LPS alters the distribution of TJprotein

Regarding the clinical treatment of Salmonellosis con-servative and supportive care remains the principal standardof management Antibiotics are only reserved for invasivediseases and patients with toxic manifestations Routineantibiotics usage appears to increase the risk of emergence ofmultidrugs resistant S typhimurium strains [6] The patientsat risk of developing multidrug resistant bacteria would ben-efit from prophylactic therapy in the past decade Howeverlack of an effective antibiotic for treatment might promotemultidrugs resistant bacteria resulting in excess morbidityand mortality [7]

The potential improvements by using probiotics inintestinal epithelial barrier function after Salmonella infec-tion have been demonstrated increasingly both in clinicaltrials and experimental models In animal models probi-otic mixtures had been used to ameliorate diarrhoea in Styphimurium-infected pigs [8] Oral administration of VSL3was also shown to be effective as a primary therapy in miceThose probiotic mixtures were thought to have an effect inenhancing barrier integrity by directly altering the epithelialpermeability and thus protecting the epithelial cells frompathogenic bacterial invasion [7]

We hypothesize that Lactobacillus is able to regulate theTJ protein expression and distribution in inflamed intestinalepithelial cells and hence change TJ structure In our previousstudy [5] we tested the interaction of S typhimurium LPS ina cultured polarized human epithelial cell modelWe success-fully demonstrated that S typhimurium LPS could disrupt TJstructure in epithelial monolayer while examining changesin resistance and cell permeability We also investigated TJprotein expression such as zonula-occludens- (ZO-) 1 actingas adapters at the cytoplasmic surface of TJ [9] as well asthe in vitro effects of Lactobacilli on TJ protein distributionOur findings suggested an important role of Lactobacillus inregulating the structure and function of TJ in intestinal cellsIn this study we tried to determine the potential abilities ofvarious Lactobacillus strains in enforcing the epithelial cellbarrier in response to the enteric pathogen S typhimuriumLPS challenge

2 Materials and Methods

21 Bacteria and Culture Conditions Lactobacillus rham-nosus GG (LGG) was purchased from BCRC (BioresourceCollection and Research Center Taiwan) The commercialstrain L casei variety rhamnosus Lcr35 (Antibiophilus) wasused in this study L rhamnosus (LR) L paracasei (LP)L johnsonii 50 (LJ50) and L johnsonii 59 (LJ59) wereobtained from TTY Biopharm (Taiwan) Those Lactobacilliwere grown under limited aeration at 37∘C in MRS medium(Difco) The number of live bacteria colony forming unit(CFU) was deduced from the absorbance at 600 nm (A600)using a calibration curve for each strain Bacterial cells weregrown till stationary phase washed twice in sterile phosphatebuffered saline (PBS pH 72) resuspended at 1 times 109 CFUmLin PBS containing 20 glycerol and stored at minus80∘C untilfurther use

22 Cell Culture and Caco-2Human Blood Peripheral Mono-cyte Cells (PBMC) CocultureModel Human colon adenocar-cinoma Caco-2 cells (Bioresource Collection and ResearchCenter BCRC 67001 Taiwan passage 33ndash37) were grown in08mL Dulbeccorsquos modified Eagle medium (DMEM) sup-plemented with 20mM L-glutamine 01mM nonessentialamino acids 10mM NaHCO

3 17mM glutamine 10mM

sodium pyruvate and 20 (vv) fetal bovine serum (ASFCBioscience Australia) Cells were seeded 1 times 105 cellscm2 in12-well transwell inserts (CORNING 04 120583m) PBMCs wereused from at least three blood donors and isolated by usingFicoll-Hypaque (GE Healthcare Bio-Science AB Sweden)gradient centrifugation from healthy donors PBMCs were(40 times 106) transferred to tissue culture plates and werecultured in 1mL RPMI 1640 containing 10 serum Cellcultures were maintained at 37∘C in 95 air and 5 CO

2

in a humidified atmosphere with three cell culture mediumchanges per week

23 Induction Inflammatory Response and Anti-InflammationScoring To induce the inflammatory response we added StyphimuriumLPS (10 ngmL L6143 Sigma) to the apical com-partment on the basis of preliminary time-course studies [5]Negative control group had no LPS treatment After 3 hr ofstimulation of positive control group all of the culture mediain both compartments were removed and washed twice withcold PBS Inflamed polarized Caco-2 monolayer was addedwith fresh medium containing various Lactobacillus strainsor positive control group without Lactobacillus for 1 6 and24 hr

24 Electrical ResistanceMeasurements Confluencewas con-trolled by measurement of the transepithelial electrical resis-tance (TEER World Precision Instruments Sarasota FLUSA) Cut-off point of TEER was defined as 450Ω times cm2and we also visualized the cell layer integrity under themicroscope for consistency TEER was read at four timepoints (0 1 6 and 24 hr)

Gastroenterology Research and Practice 3

25 Immunofluorescence Microscopy For immunofluores-cence study polarized Caco-2 monolayers were plated ontochamber glass slides (Deckglaser) andwashed twice with PBS3 hr prior to the exposure to LPS (10 ngmLminus1) Lactobacilluswas added into the inflamed monolayers and incubated for24 hrs Cells were then fixed in 4paraformaldehyde (Merck)and permeabilized with 03 Triton X-100 (Sigma-Aldrich)To study the TJ unspecific bindings were blocked with 5fetal bovine serum (SAFC Australia) 45min prior to theincubation with primary antibody (1 300 mouse anti-tightjunction protein-1 (anti-ZO-1) 01mgmL Sigma USA) for60min and secondary antibody (1 500 Dylight 488 goatantirabbit immunoglobulin G Jackson USA) for 30minTheresults were analyzed using a BX60 fluorescence microscope(Olympus Hamburg Germany) ZO-1 staining was observedblindly by two individual observers in duplicate samples andperformed three times

26 Western Blot Inflamed polarized Caco-2 monolayerswere added Lactobacillus and grown on 24-wells plate Theywere harvested after incubation for 24 hr Individual sampleswere lysed in an ice-cold lysis buffer (20mM Tris-HCl pH74 150mMNaCl 5mM MgCl

2 10 glycerol 05 NP-40

01 SDS 03 120583M aprotinin 1 120583M leupeptin 1mM PMSF1 120583M pepstatin A) and placed on ice for 10 minutes Thelysed samples were centrifuged at 10000 rpm for 5 minutesat 4∘C and the supernatant was collected Total proteinconcentration was quantified by use of the BCA protein assaykit (Thermo USA) For protein analysis 40120583g of protein wasadded to an equal volume of 2 times Laemmli sample buffer andboiled for 10 minutes The samples were run at 8 poly-acrylamide gel at 100V for 15 hr Protein was transferred toImmunoblot PVDF membranes (Bio-Rad) After overnightblocking (PBSTween supplemented with 005 nonfat drymilk) blots were incubated with primary (mouse anti-tightjunction protein-1 (anti-ZO-1) (Sigma USA)) and secondaryantibodies (horseradish peroxidase conjugated anti-mouseIgG (Jackson USA)) for 60 minutes at room temperatureproteins were visualized by chemiluminescence reagents(ThermoUSA) and exposed to X-OMATfilm (Kodak USA)

Statistics The quantitative data were expressed as mean plusmnstandard error (SE) for triplicate measurements Statisticalanalyses were performedwith Studentrsquos t-test using SPSS 120Statistical significance was defined as a P value of lt005

3 Results

31 Effect of Lactobacillus onCaco-2Monolayer Resistance Todeterminewhether the alterations inCaco-2monolayer resis-tance required the direct effects between the Lactobacillusand intestinal epithelial cells we selected six strains of Lac-tobacillus and exposed them to polarized Caco-2 monolayerindividually Significant differences in the measured valuesfor the polarized monolayer were observed 3 hr after LPStreatment TEER was a measurement of the integrity of theepithelial barrier andwasmonitored over the course of a 24 hr

60

80

100

120

140

160

180

0 6 12 18 24

Ratio

of

TEER

()

(h)

Positive controlLRLPLJ50

LJ59LGGLcr35

lowastlowastlowastlowast

lowastlowast

lowast

lowast

Figure 1 Effect of Lactobacilli on Caco-2 monolayer resistanceTEER ratio of inflamed Caco-2 monolayers exposed to positivecontrol medium (◼) LGG (998771) Lcr35 (◻) LR () LP (∙) LJ50 (X)and LJ59 () into the apical compartment TEER (Ω times cm2) wasexpressed as the percentage at time 3 hr in relation to the initial valuefor each treatment lowast119875 lt 005 lowastlowast119875 lt 0005

protection We found that the TEER values from culturedepithelial cells in positive control group were consistentlythe lowest at each time frame (Figure 1) TEER values ofvarious Lactobacillus groups remained relatively close 1 hrafter incubation Most Lactobacillus strains did not induce asignificant increase of TEER 6 hr later However after 24 hrincubation TEER values in LP and LR groups were found tohave the highest levels among all Lactobacillus groups TEERlevels of Lcr35 group remained the lowest through the 24 hrperiod TEER values in LGG LJ50 and LJ59 groups werelower than those of LP and LR groups and had statisticallysignificant differences These results suggested that variousstrains of Lactobacillus were responsible for the differentmodulations of cell layer integrity

32 Effects of LPS on ZO-1 Location and Expression AsTJwascrucial for the barrier function the effects of Lactobacilluson the TJ integrity were further studied To determine theexpression of ZO-1 on LPS-induced disruption of TJ weperformed the Western blot analysis and found that theTJ membrane protein ZO-1 was markedly degraded in PCand PC-24 h groups (Figure 2(a)) We also used TJ markerincluding ZO-1 for immunofluorescence microscopy studyon colon epithelium (Figure 2(b)) ZO-1 distribution inthe negative control group cells had its normally smoothnature However ZO-1 was highly disrupted in epithelialcells exposed to LPS appearing in a discontinuous bead-like pattern in the LPS treatment group The 3 hr exposureto LPS resulted in loss of ZO-1 at the intercellular junctionscompared to the negative control group LPS was found to be


LJ59

Lcr3

5

LR LJ50

LP LGG

GAPDH

ZO-1

Con

trol

180 kDa

38kDa

LPS3

hr

LPS24

hr

(a)

NC

PC

LGG

LR

Lcr35

LJ59

LJ50

LP

10120583m

10120583m

10120583m10120583m10120583m

10120583m 10120583m

10120583m10120583m

PC-24h

(b)

Figure 2 The appearances of tight junctions were evinced by immunostaining ZO-1 expression and localization after LPS and Lactobacillusadministration Negative control (NC) without treatment and positive control (PC) exposed to LPS for 3 hr were shown Inflamed Caco-2cells were colonized without bacteria (PC-24 h) or with LGG LR LP Lcr35 LJ50 or LJ59 for 24 hr (a) Representative 180-kDa ZO-1 bandwas shown by Western blot analyses Expression of ZO-1 showed significantly lower levels in the PC and PC-24 h groups when compared tothe NC group (b) Representative slides were evaluated by immunofluorescence microscopy Expression of ZO-1 showed similar levels in theLR LP and LGG groups when compared to the NC group GAPDH glyceraldehyde 3-phosphate dehydrogenase

involved in the disruption of epithelial barrier and disruptedthe location of ZO-1

33 Effects of Lactobacillus on ZO-1 Location and ExpressionExposed to LPS Lactobacillus plays an important role inthe maintenance of epithelial barrier [1] Our Western blotanalysis revealed a significant increase of ZO-1 expressionin various Lactobacillus strains and showed similar densitiesexcept in PC PC-24 h and LJ59 groups (Figure 2(a)) Inaddition expression of ZO-1 showed similar levels in the LRLP and LGG groups when compared to the NC group Wefound that these Lactobacilli strains were also responsible forthe changes in the location of TJ protein Polarized Caco-2 monolayers exposed to LPS were incubated with various

strains of Lactobacillus for 24 hr and the localization of ZO-1 was examined via microscopy (Figure 2(b)) The disruptedTJ of negative control group could not be reversed after24 hr incubation In negative control strains of LGG andLcr35 had no effect on TJ structure appearance and sizeBesides exposure to LR also caused the TJ structure tocurve markedly and ZO-1 became larger in appearance Innegative control and LP-incubated cells groups ZO-1 wasfound to be localized to the lateral cell membrane and formeda characteristic continuous web-like pattern Moreover wefound cloudy web-like ZO-1 appearance in the groups withstrains LJ 50 and LJ 59 treatments Even after the removalof LPS for 24 hr we found the appearance of ZO-1 remainedthe same As a conclusion we showed here that the disrupted


TJ could not be reversed at limited period after the removalof LPS However lactic acid bacteria strains could be able toconserve the appearance of the TJ entirely

4 Discussion

In human beings the mucosal barrier of the gastrointestinaltract is composed of the intestinal microbial flora the mucuslayer the epithelial cells and the intercellular TJ positionedbetween them [10] TJ are the key molecules involved in thecontrol of paracellular permeability [11 12] TJ are complexprotein structures comprised of transmembrane proteinswhich interact with the actin cytoskeleton via plaque pro-teins Signaling pathways involved in the assembly disas-sembly and maintenance of TJ are controlled by a numberof signaling molecules such as protein kinase C mitogen-activated protein kinases myosin light chain kinase andRho GTPases [13] They seal the paracellular space betweenepithelial cells thus preventing paracellular diffusion ofmicroorganisms and other antigens across the epithelium

Commensal bacteria which normally colonize themurine gut during the first several weeks of postnatal lifeinduce expression of genes that improve intestinal barrierfunction whereas abnormal bacterial colonization maydisrupt this process and contribute to the development ofhost diseases [14] Commensal probiotics have been shownto promote intestinal barrier integrity both in vitro and invivo Probiotics preserve the intestinal barrier in mousemodels of colitis [8] and reduce intestinal permeabilityin human patients with Crohnrsquos Disease [15] Treatmentof epithelial cells with Escherichia coli Nissle 1917 leads toincrease in expression of ZO-2 protein and redistributionof ZO-2 from the cytosol to cell boundaries in vitro [16]Recently L plantarum DSM 2648 was able to reduce thenegative effect of E coli (enteropathogenic E coli (EPEC))O127H6 (E234869) on TEER and adherence by as muchas 9875 and 8018 respectively during simultaneousor prior coculture compared with EPEC incubation alone[17] Administration of L plantarum into the duodenumof healthy human volunteers was shown to significantlyincrease ZO-1 and occludin in the vicinity of TJ structures[18]

In this study we showed that Lactobacillus could attenuatethe barrier disruption disruption of intestinal epithelial cellscaused by Salmonella LPS administration Addition of Lac-tobacillus to the cell culture medium was able to reduce theLPS-induced inhibition of TEER and reverse the change inTJ protein ZO-1 expression

Although this present study did not address the mech-anism by which probiotics inhibited LPS-induced damagein Caco-2 cells it could be hypothesized that E coli strainNissle 1917 (EcN) might do so by restoration of a disruptedepithelial barrier infected by E coli strain E234869 [16]Other studies indicated that the anti-inflammatory effectof live Lactobacillus acidophilus on increased transepithelialresistance contrasts markedly with the fall in resistanceevoked by E coli infection [19] The fact that different

Lactobacillus strains had different varying characteristics inrepairing the intestinal barrier might explain why LP wasable to recover the LPS-induced damage in polarized Caco-2monolayer more efficiently than LJ50 and LJ59 did when welooked at their TEER expressions

Another conceivable mode of action for lactic acid bacte-ria is that they directly modulated the function of epithelialcells The TJ might play a role in preventing the entranceof pathogens into the epithelial cells It was reported thatfermented fruit by probiotics stimulates TJ maintenance andformation [20] In addition LGG had the ability to preventchanges in host cell morphology attachingeffacing lesionformation and monolayer resistance against enterohemor-rhagic E coliO157H7 infection [21] At least some probioticswere shown to have the ability of stabilizing TJ and inducingmucin secretion in epithelial cells [22] After LPS stimulationcoincubation with strain LC could stabilize TJ structure aswell Besides the addition of strains LGG and Lcr35 alsoresulted in a similar TJ morphology as original in the Caco-2 monolayer In contrast to the LJ59 group TJ structure wasmore poorly maintained compared to positive control Thuswe found Lactobacillus may have the potential to protect theepithelial barrier from damage induced by LPS direct actionon the epithelial cells

Although in different study settings previous studiesreported that Lactobacillus had beneficial anti-inflammatoryeffects on Salmonella [5] it is still difficult to ascertain theirdirect mechanism of action Our previous findings docu-mented the anti-inflammatory effects of Lcr35 SalmonellaLPS [5] Several studies showed that administration of dif-ferent Lactobacillus strains could reduce diarrhea [23] Dam-aged tight junction and disrupted intestinal barrier occurduring enterocolitis In addition probiotics administrationhad shown to have an anti-inflammatory effect and play animportant role in treatment of enterocolitis [24]

In conclusion we provide insight that live probioticscould improve epithelial barrier properties and this mayexplain the potential mechanism behind their beneficialeffect in vivo

Conflict of Interests

The authors declare no conflict of interests in relation tothis paper The authors declare no conflict of ethics inrelation to this paper The authors alone are responsiblefor the content and writing of the paper The authors haveneither a commercial nor other associations that might posea conflict of interest (eg pharmaceutical stock ownershipconsultancy advisory boardmembership relevant patents orresearch funding)

Disclosure

Part of information has been present in the 43rd AnnualMeeting of The European Society for Paediatric Gastroen-terology Hepatology andNutrition June 9ndash12 2010 IstanbulTurkey The abstract number is PO-N-414


Acknowledgment

This study was supported in part by a research Grantfrom theMackayMemorial Hospital (MMH-E-101-12-1)Theauthors thank TTY Biopharm (Taiwan) for providing theLactobacillus strains

References

[1] R C Anderson A L Cookson W C McNabb W J Kelly andN C Roy ldquoLactobacillus plantarum DSM 2648 is a potentialprobiotic that enhances intestinal barrier functionrdquo FEMSMicrobiology Letters vol 309 no 2 pp 184ndash192 2010

[2] Y H Bai S C Pak S H Lee et al ldquoAssessment of a bioactivecompound for its potential antiinflammatory property by tightjunction permeabilityrdquo Phytotherapy Research vol 19 no 12 pp1009ndash1012 2005

[3] G Barbara L Zecchi R Barbaro et al ldquoMucosal permeabilityand immune activation as potential therapeutic targets ofprobiotics in irritable bowel syndromerdquo Journal of ClinicalGastroenterology vol 46 pp S52ndashS55 2012

[4] L S Bertelsen G Paesold S L Marcus B B Finlay LEckmann and K E Barrett ldquoModulation of chloride secretoryresponses and barrier function of intestinal epithelial cellsby the Salmonella effector protein SigDrdquo American Journal ofPhysiology vol 287 no 4 pp C939ndashC948 2004

[5] H W Fang S B Fang J S C Chiau et al ldquoInhibitoryeffects of Lactobacillus casei subsp rhamnosus on Salmonellalipopolysaccharide-induced inflammation and epithelial bar-rier dysfunction in a co-culture model using Caco-2peripheralblood mononuclear cellsrdquo Journal of Medical Microbiology vol59 no 5 pp 573ndash579 2010

[6] J H Brumell O Steele-Mortimer and B B Finlay ldquoBacterialinvasion force feeding by Salmonellardquo Current Biology vol 9no 8 pp R277ndashR280 1999

[7] N F Crum-Cianflone ldquoSalmonellosis and the gastrointestinaltract more than just peanut butterrdquo Current GastroenterologyReports vol 10 no 4 pp 424ndash431 2008

[8] P G Casey G E Gardiner G Casey et al ldquoA five-strain pro-biotic combination reduces pathogen shedding and alleviatesdisease signs in pigs challengedwith Salmonella enterica serovarTyphimuriumrdquo Applied and Environmental Microbiology vol73 no 6 pp 1858ndash1863 2007

[9] E C Boyle N F Brown and B B Finlay ldquoSalmonella entericaserovar Typhimurium effectors SopB SopE SopE2 and SipAdisrupt tight junction structure and functionrdquo Cellular Micro-biology vol 8 no 12 pp 1946ndash1957 2006

[10] S Guandalini ldquoProbiotics for prevention and treatment ofdiarrheardquo Journal of Clinical Gastroenterology vol 45 pp S149ndashS153 2011

[11] P Gupta H Andrew B S Kirschner and S Guandalini ldquoIsLactobacillus GG helpful in children with Crohnrsquos diseaseResults of a preliminary open-label studyrdquo Journal of PediatricGastroenterology and Nutrition vol 31 no 4 pp 453ndash457 2000

[12] M A Jepson H B Schlecht and C B Collares-Buzato ldquoLocal-ization of dysfunctional tight junctions in Salmonella entericaserovar typhimurium-infected epithelial layersrdquo Infection andImmunity vol 68 no 12 pp 7202ndash7208 2000

[13] 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-induced

changes in epithelial barrier functionrdquo Infection and Immunityvol 76 no 4 pp 1340ndash1348 2008

[14] J Karczewski F J Troost I Konings et al ldquoRegulation ofhuman epithelial tight junction proteins by Lactobacillus plan-tarum in vivo and protective effects on the epithelial barrierrdquoAmerican Journal of Physiology vol 298 no 6 pp G851ndashG8592010

[15] L Khailova K Dvorak K M Arganbright et al ldquoBifidobac-terium bifidum improves intestinal integrity in a rat model ofnecrotizing enterocolitisrdquo American Journal of Physiology vol297 no 5 pp G940ndashG949 2009

[16] 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

[17] J Miyoshi and Y Takai ldquoMolecular perspective on tight-junction assembly and epithelial polarityrdquo Advanced DrugDelivery Reviews vol 57 no 6 pp 815ndash855 2005

[18] A Nusrat J R Turner and J L Madara ldquoMolecular physiologyand pathophysiology of tight junctions IV Regulation of tightjunctions by extracellular stimuli nutrients cytokines andimmune cellsrdquo American Journal of Physiology vol 279 no 5pp G851ndashG857 2000

[19] J M Otte and D K Podolsky ldquoFunctional modulation of ente-rocytes by gram-positive and gram-negative microorganismsrdquoAmerican Journal of Physiology vol 286 no 4 pp G613ndashG6262004

[20] R M Patel L S Myers A R Kurundkar A Maheshwari ANusrat and P W Lin ldquoProbiotic bacteria induce maturation ofintestinal claudin 3 expression and barrier functionrdquo AmericanJournal of Pathology vol 180 pp 626ndash635 2012

[21] 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

[22] J R Turner ldquoIntestinal mucosal barrier function in health anddiseaserdquoNature Reviews Immunology vol 9 no 11 pp 799ndash8092009

[23] D Ulluwishewa R C AndersonW CMcNabb P J MoughanJ M Wells and N C Roy ldquoRegulation of tight junctionpermeability by intestinal bacteria and dietary componentsrdquoJournal of Nutrition vol 141 no 5 pp 769ndash776 2011

[24] 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 andPKC120577 redistribution resulting in tight junction and epithelialbarrier repairrdquo Cellular Microbiology vol 9 no 3 pp 804ndash8162007

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synergistically with other conditions such as inflammatoryresponses to promote tight junction (TJ) dysfunction [4]At present S typhimurium overcoming the intestinal barrieris the most widely accepted mechanism for their enteringinto host cells especially deeper tissues Thus the mainte-nance of the tight junction integrity of polarized epithelialmonolayer plays an important role in avoiding bacterialinvasion

Besides the disrupted barrier function by S typhimuriumeffectors evidence also showed that lipopolysaccharide (LPS)could induce derangements in intestinal epithelial barrierfunction In our previous study we demonstrated the evi-dence of Salmonella LPS-induced inflammation and epithe-lial barrier dysfunction [5] However the specific TJ respon-sible for the barrier dysfunction has not been identified Byusing polarized epithelial monolayers we might clarify themechanism that Salmonella LPS alters the distribution of TJprotein

Regarding the clinical treatment of Salmonellosis con-servative and supportive care remains the principal standardof management Antibiotics are only reserved for invasivediseases and patients with toxic manifestations Routineantibiotics usage appears to increase the risk of emergence ofmultidrugs resistant S typhimurium strains [6] The patientsat risk of developing multidrug resistant bacteria would ben-efit from prophylactic therapy in the past decade Howeverlack of an effective antibiotic for treatment might promotemultidrugs resistant bacteria resulting in excess morbidityand mortality [7]

The potential improvements by using probiotics inintestinal epithelial barrier function after Salmonella infec-tion have been demonstrated increasingly both in clinicaltrials and experimental models In animal models probi-otic mixtures had been used to ameliorate diarrhoea in Styphimurium-infected pigs [8] Oral administration of VSL3was also shown to be effective as a primary therapy in miceThose probiotic mixtures were thought to have an effect inenhancing barrier integrity by directly altering the epithelialpermeability and thus protecting the epithelial cells frompathogenic bacterial invasion [7]

We hypothesize that Lactobacillus is able to regulate theTJ protein expression and distribution in inflamed intestinalepithelial cells and hence change TJ structure In our previousstudy [5] we tested the interaction of S typhimurium LPS ina cultured polarized human epithelial cell modelWe success-fully demonstrated that S typhimurium LPS could disrupt TJstructure in epithelial monolayer while examining changesin resistance and cell permeability We also investigated TJprotein expression such as zonula-occludens- (ZO-) 1 actingas adapters at the cytoplasmic surface of TJ [9] as well asthe in vitro effects of Lactobacilli on TJ protein distributionOur findings suggested an important role of Lactobacillus inregulating the structure and function of TJ in intestinal cellsIn this study we tried to determine the potential abilities ofvarious Lactobacillus strains in enforcing the epithelial cellbarrier in response to the enteric pathogen S typhimuriumLPS challenge

2 Materials and Methods

21 Bacteria and Culture Conditions Lactobacillus rham-nosus GG (LGG) was purchased from BCRC (BioresourceCollection and Research Center Taiwan) The commercialstrain L casei variety rhamnosus Lcr35 (Antibiophilus) wasused in this study L rhamnosus (LR) L paracasei (LP)L johnsonii 50 (LJ50) and L johnsonii 59 (LJ59) wereobtained from TTY Biopharm (Taiwan) Those Lactobacilliwere grown under limited aeration at 37∘C in MRS medium(Difco) The number of live bacteria colony forming unit(CFU) was deduced from the absorbance at 600 nm (A600)using a calibration curve for each strain Bacterial cells weregrown till stationary phase washed twice in sterile phosphatebuffered saline (PBS pH 72) resuspended at 1 times 109 CFUmLin PBS containing 20 glycerol and stored at minus80∘C untilfurther use

22 Cell Culture and Caco-2Human Blood Peripheral Mono-cyte Cells (PBMC) CocultureModel Human colon adenocar-cinoma Caco-2 cells (Bioresource Collection and ResearchCenter BCRC 67001 Taiwan passage 33ndash37) were grown in08mL Dulbeccorsquos modified Eagle medium (DMEM) sup-plemented with 20mM L-glutamine 01mM nonessentialamino acids 10mM NaHCO

3 17mM glutamine 10mM

sodium pyruvate and 20 (vv) fetal bovine serum (ASFCBioscience Australia) Cells were seeded 1 times 105 cellscm2 in12-well transwell inserts (CORNING 04 120583m) PBMCs wereused from at least three blood donors and isolated by usingFicoll-Hypaque (GE Healthcare Bio-Science AB Sweden)gradient centrifugation from healthy donors PBMCs were(40 times 106) transferred to tissue culture plates and werecultured in 1mL RPMI 1640 containing 10 serum Cellcultures were maintained at 37∘C in 95 air and 5 CO

2

in a humidified atmosphere with three cell culture mediumchanges per week

23 Induction Inflammatory Response and Anti-InflammationScoring To induce the inflammatory response we added StyphimuriumLPS (10 ngmL L6143 Sigma) to the apical com-partment on the basis of preliminary time-course studies [5]Negative control group had no LPS treatment After 3 hr ofstimulation of positive control group all of the culture mediain both compartments were removed and washed twice withcold PBS Inflamed polarized Caco-2 monolayer was addedwith fresh medium containing various Lactobacillus strainsor positive control group without Lactobacillus for 1 6 and24 hr

24 Electrical ResistanceMeasurements Confluencewas con-trolled by measurement of the transepithelial electrical resis-tance (TEER World Precision Instruments Sarasota FLUSA) Cut-off point of TEER was defined as 450Ω times cm2and we also visualized the cell layer integrity under themicroscope for consistency TEER was read at four timepoints (0 1 6 and 24 hr)







3 Results


60

80

100

120

140

160

180

0 6 12 18 24

Ratio

of

TEER

()

(h)


LJ59LGGLcr35


lowastlowast

lowast

lowast





LJ59

Lcr3

5

LR LJ50

LP LGG

GAPDH

ZO-1

Con

trol

180 kDa

38kDa

LPS3

hr

LPS24

hr

(a)

NC

PC

LGG

LR

Lcr35

LJ59

LJ50

LP

10120583m

10120583m

10120583m10120583m10120583m

10120583m 10120583m

10120583m10120583m

PC-24h

(b)







4 Discussion











Disclosure



Acknowledgment


References































of






Disease Markers



OncologyJournal of





PPAR Research



Journal of

ObesityJournal of






















3 Results


60

80

100

120

140

160

180

0 6 12 18 24

Ratio

of

TEER

()

(h)


LJ59LGGLcr35


lowastlowast

lowast

lowast





LJ59

Lcr3

5

LR LJ50

LP LGG

GAPDH

ZO-1

Con

trol

180 kDa

38kDa

LPS3

hr

LPS24

hr

(a)

NC

PC

LGG

LR

Lcr35

LJ59

LJ50

LP

10120583m

10120583m

10120583m10120583m10120583m

10120583m 10120583m

10120583m10120583m

PC-24h

(b)







4 Discussion











Disclosure



Acknowledgment


References































of






Disease Markers



OncologyJournal of





PPAR Research



Journal of

ObesityJournal of

















LJ59

Lcr3

5

LR LJ50

LP LGG

GAPDH

ZO-1

Con

trol

180 kDa

38kDa

LPS3

hr

LPS24

hr

(a)

NC

PC

LGG

LR

Lcr35

LJ59

LJ50

LP

10120583m

10120583m

10120583m10120583m10120583m

10120583m 10120583m

10120583m10120583m

PC-24h

(b)







4 Discussion











Disclosure



Acknowledgment


References































of






Disease Markers



OncologyJournal of





PPAR Research



Journal of

ObesityJournal of


















4 Discussion











Disclosure



Acknowledgment


References































of






Disease Markers



OncologyJournal of





PPAR Research



Journal of

ObesityJournal of

















Acknowledgment


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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