Infectious diarrhoea caused by bacterial entericpathogenscontinuestobeamajorcauseofmorbidityand childhood mortality in developing countries1.Whileadaptiveimmunityoccursonexposuretotheseentericbacterialpathogens, innate immuneresponses

Effectsofenteropathogenicbacteria&lactobacillionchemokinesecretion&Tolllikereceptorgeneexpressionintwohumancolonicepithelialcelllines

N.S.Nandakumar,S.Pugazhendhi&B.S.Ramakrishna

Department of Gastrointestinal Sciences, Christian Medical College, Vellore, India

ReceivedApril13,2008

Background & objectives: The intestinal epithelium is part of the innate immune system responding to contact with pathogenic or commensal bacteria. The objective of this study was to compare innate responses of intestinal epithelial cell lines to pathogenic bacteria and to lactobacilli.

Methods: Two human intestinal epithelial cell lines, HT29 (enterocyte-like) and T84 (crypt-like), were exposed to pathogenic bacteria representative of non invasive (Vibrio cholerae O1 and O139), adherent (enterohaemorrhagic Escherichia coli, EHEC) or invasive (Salmonella Typhimurium and Shigella flexneri) phenotypes and to non pathogenic Lactobacillus rhamnosus GG or Lactobacillus plantarum. Interleukin-8 (IL-8) was measured in culture supernatant by ELISA, while mRNA from cells was subjected to quantitative reverse transcriptase PCR for several other chemokines (CXCL1, CCL5 and CXCL5) and for Toll-like receptors (TLR) 2, 4, 5 and 9.

Results: V. cholerae, S. Typhimurium, S. flexneri and EHEC induced IL-8 secretion from epithelial cells into the medium. Salmonella, Shigella and EHEC, but not V. cholerae, significantly increased mRNA expression of CXCL1. None of the pathogens induced CCL5 or CXCL5. Salmonella and Vibrio significantly increased TLR4 expression, while Vibrio and EHEC decreased TLR5 expression. EHEC also decreased TLR9 expression. Lactobacilli attenuated the IL-8 response of the cell lines to V. cholerae, Salmonella, and EHEC but did not significantly change the IL-8 response to Shigella.

Interpretation & conclusions: Distinct patterns of epithelial cell chemokine responses were induced by the bacterial pathogens studied and these were modulated by commensal lactobacilli. Alterations in TLR expression by these pathogens are likely to be important in pathogenesis.

Key words Chemokine-enteropathogenicbacteria-interleukin-8-intestinalepithelialcells-Lactobacillus-Toll-likereceptor

areincreasinglyrecognizedasimportanttobothdiseasepathogenesis and immunity2. Bacterial pathogenscausediarrhoeainvariousways.SomesuchasVibrio choleraecolonizethebowel,produceenterotoxins,andinduce fluid secretion and diarrhoea, without invading

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the epithelium or mucosa. Others such as Shigellaspeciesinvadetheepitheliumleadingtoepithelialcelldeath and ulceration and causing blood and mucusdiarrhoea. Enterohaemorrhagic Escherichia coli(EHEC) adhere to epithelial brush border membraneand secrete a cytotoxin. The intestinal epithelial cellis the first cell type that comes into contact with the bacteriarecognizingthemthroughpatternrecognitionreceptors, principally the Toll-like receptors (TLRs),locatedonthecellsurface3.Thiscontacttriggersreleaseof inflammatory mediators, the most prominent of which is interleukin-8 (IL-8) a neutrophil chemotacticpeptide4-6. Other pro-inflammatory mediators include growth-related oncogene-α (GRO-α, now known asCXCL1)andepithelialcell-derivedneutrophilactivatingfactor(ENA-78,alsoknownasCXCL5)7,bothofwhicharechemotactictoneutrophils.ExpressionofRANTES(regulateduponactivation,normalT-cellexpressedandsecreted, CCL-5), which is chemotactic to T cells, isincreasedinrotavirus-infectedepithelialcells8.

MammalianTLRsplayacrucialdual role inhostimmunity9,10andareimportantfor thedevelopmentofhostinnateandadaptiveimmuneresponsestogastrointestinalinfections.TLR2that recognizespeptidoglycanmotifsof bacterial cell walls, and TLR4 that recognizeslipopolysaccharide may be present in epithelial cells,but aremore likely tobe found in sub-epithelial cellsincluding myofibroblasts10. TLR5 that recognizesflagellin, the primary structural component of bacterial flagella, is possibly responsible for pro-inflammatory gene activation in epithelial cells in response topathogenicbacteria11.Interestingly,acommensalE. colistrainhasalsobeenshowntoactivateTLR5inintestinalepithelialcells12.TLR9,thatrecognizesunmethylatedCpGDNA,mediates the downregulation of inflammatory gene activationbycommensallactobacilliinananimalmodelof inflammatory bowel disease13.

Lactobacilli are normal commensal flora of the gut thatareusedinthetherapyofdiarrhoeaandintestinalinflammation. Lactobacillus rhamnosus GG (LGG),one of the most widely studiedprobiotic bacteria, iseffectiveinpreventingandtreatingdiarrhoeaininfancyand childhood14, and has a cytoprotective effect onintestinal epithelial cells15.Other lactobacilli are alsoused as probiotics, in particularL. plantarum,whichiswidelyused in clinical practice16.Previous studieshaveshownthatlactobacilliinhibitgrowthofseveralpathogens including V. cholerae17,18. Lactobacilli canalso inhibit Shigella-induced nuclear factor kappa B(NFκB) activation in epithelial cells19. However, the

effectsoflactobacilliontheinteractionbetweenentericbacterial pathogens and epithelial cells (in particularwhetherprobiotic:pathogenratioisimportant,whetherthese exert a protective effect if administered eitherprior to or simultaneously with pathogen, and acomparisonofprobioticeffectsagainstthebackgroundofmechanismusedtocausediarrhoea)hasneverbeenexamined. Since epithelial cell interactions occur todifferent degrees and through different mechanismswith the various bacterial enteric pathogens, it ispossiblethattheinnateimmuneresponsesinducedinepithelial cells vary in type and degree. The presentstudies were designed to compare the intestinalepithelial cell responses to different enteric bacterialpathogens, to determine the influence of lactobacilli ontheresponsesinducedbytheentericpathogens,andto determine whether any of the pathogenic bacteriacausedalterationsinexpressionofmRNAforTLRsofinterestincolonicepithelialcelllines.

Material & Methods

Bacteria:ClinicalisolatesofSalmonella Typhimurium, S. flexneri, enterohaemorrhagic E. coli (EHEC),V. choleraeO1 andV. choleraeO139, cultured frompatients with gastroenteritis, were used for thesestudies.Thesebacteriawerechosenasrepresentativeofdifferenttypesofpathogenesis.Salmonella andShigellaare invasive pathogens, EHEC adhere to epithelialcell surfaces, and V. cholerae are non invasive, nonadherent,toxin-producingbacteria.L. rhamnosus GG(LGG)wasgrown fromCulturelle capsulesobtainedcommercially and L. plantarum was obtained fromNational Chemical Laboratory (NCL), Pune, India. Lactobacilli were grown in de Man Rogosa Sharpebroth(Hi-media,India)underanaerobicconditionsfor16hat37°Conthedaybeforetheexperiments.

Cell lines:Twohumancoloncarcinomacelllineswereusedinthesestudies.TheHT29cellline[obtainedfromNationalCentreforCellScience(NCCS),Pune,India]hasanenterocyte-likedifferentiationandisconsideredtoberepresentativeofsurfaceorvillustypecells.TheT84cells(ATCC,USA)haveasecretorydifferentiationandareconsideredrepresentativeofthesecretorycellsthat linethebottomoftheintestinalorcoloniccrypts.HT29cellsweregrownin25cm2 flasks (Axygen, India) in high glucose Dulbecco’s Modified Eagle Medium (DMEM) (GIBCO Invitrogen, India) supplementedwith10per cent foetal bovine serum (GIBCO, India)containing penicillin (100 U/ml), streptomycin (100µg/ml) and amphotericin B (10 µg/ml) at 37°C with

NANDAKUMARet al:ENTERICBACTERIA&EPITHELIALTOLL-LIKERECEPTOR&CHEMOKINEEXPRESSION 171

5percentCO27.T84cellswereculturedinDMEMand

Ham’sF12medium(GIBCO,India)containingpenicillin(100U/ml),streptomycin(100µg/ml)andamphotericinB(10µg/ml)at37°Cwith5percentCO2

7.

Bacteria-cell interactions: Bacteria were grownstandingovernightat37°CinLuria–Bertani(L-B)broth(Hi-media,India)tologphasebeforeuse,pelletedbycentrifugationat500g for10min,andwashedthreetimeswithsterilephosphatebufferedsaline(pH7.4).Thebacterialconcentrationinthesuspensionmediumwasdeterminedbymeasurementofabsorbanceat540-600 nm and comparison with standard MacFarlandtubes. The bacteria were resuspended in antibiotic-free medium and overlaid on epithelial cells to a final concentrationof100bacteriaperepithelialcellfortheinfection studies4-7. HT29 and T84 cell monolayerswereculturedin24-wellplates(FalconMilliwell,BD,USA) and grown until they reached confluence. Prior toinfection,cellswerewashedtwicewithappropriatemediumwithoutserumandantibioticsandmaintainedinserumfreemediumforatleast2h.Epithelialcellswere counted from a duplicate well in a Neubauerchamber.Theepithelialcellsinthewellswereoverlaidwith the test bacteria in suspension at a ratio of 100bacteria per epithelial cell and incubated at 370C for2 h. The extracellular bacteria were then removedbywashingand thecell lineswere further incubatedfor 4 h in the presence of 50 µg/ml gentamicin tokill any remaining extracellular bacteria.At the end,culturesupernatantwas removedandstoredat -200Cfor quantitation of IL-8 secretion, while cells wereremovedandstoredat-800CfortotalRNAisolation.Instudieswithlactobacilli,theappropriatebacteriawereaddedeitheraloneinordertoascertainwhethertherewas any basal effect, or added either along with orprior toenteropathogenicbacteria.Whenaddedpriortotheenteropathogen,thelactobacilliwereincubated

withthecelllinefor2h,washedandremoved,andtheenteropathogenthenadded,incubatedwiththecelllineforafurthertwohoursthenwashed,andthecelllineincubatedat370Cinpresenceofgentamicinforanother4h.Inadditiontheeffectoflactobacilliconcentrationwastestedusingtwodosesoflactobacilli(ratioof1:1or10:1lactobacillitoenteropathogen).Thelatterdosewaschosenempirically,sincethereisnoliteraturetosupport the use of a specific dose. Experiments were donethreetimes,eachtimeinduplicatewells.

IL-8 assay and quantitative PCR for chemokine and TLR mRNA:Theculturesupernatantswerecentrifugedat 12,000 g for 10 min and IL-8 was assayed byELISA (OptEIA Set, Becton Dickinson, USA). Thetranscriptional profile of several other chemokines, CXCL1, CXCL5 and CCL5, was investigated. TotalRNA was isolated from infected cells using Trizolreagent (Sigma, India) as per the manufacturer’sinstructions. Reverse transcription of RNA wasperformed as per manufacturer’s instructions (www.finnzymes.fi/pdf/F-572_guidelines.pdf) in a final volumeof20µlcontaining0.5mMofeachnucleotidetriphosphate,40unitsofRNAase inhibitor (Ambion,USA), 50-100 ng of Random Hexamers (AmershamPharmacia, UK), 200 units of Moloney MurineLeukemia Virus Reverse Transcriptase (MMuLV-RT,Finnzymes,Finland)and15µloftheextractedRNAorH2O,andthenincubatedat42°Cfor1h.Subsequently1µl of diluted cDNA sample was amplified in a thermal cycler(MJResearch,USA)in20µlof1xPCRbuffercontaining200µMofeachnucleotidetriphosphate,10picomolesofeachprimer(SigmaGenosysBangalore)(Table),andTitaniumTaqDNApolymerase(Clontech,BectonDickinson,USA).

Quantitative real-time PCR20 was performed withappropriateconditions inaChromo4system(Biorad,USA). 1µg of RNA was transcribed to cDNA using

Table. Primers used for PCR amplification of chemokines and TLRs

Gene Forward Reverse Productlength(bp)

Chemokines: CXCL1(GRO) 5’-ATGGCCCGCGCTGCTCTCT-3’ 5’-AGCTTTCCGCCCATTCTTG-3’ 251 CCL5(RANTES) 5’-TACCATGAAGGTCTCCGC-3’ 5’-GACAAAGACGACTGCTGG-3’ 198 CXCL5(ENA78) 5’-GTGTTGAGAGAGCTGCGTTG-3’ 5’-TTTTCCTTGTTTCCACCGCT-3’ 215Toll like receptors: TLR 2 5’-CAATGATGCTGCCATTCTCAT-3’ 5’-ATTATCTTCCGCAGCTTGCA-3’ 83 TLR 4 5’-AGTTTCCTGCAATGGATCAAGG-3’ 5’-CTGCTTATCTGAAGGTGTTGCAC-3’ 83 TLR 5 5’-GGCTTAATCACACCAATGTCACTAT-3’ 5’-GAAACCCCAGAGAACGAGTCAG-3’ 81 TLR 9 5’-AGTCAATGGCTCCCAGTTCCT–3’ 5’-CGTGAATGAGTGCTCGTGGTA-3’ 93House keeping gene:Beta Actin 5’-TCCCTGGAGAAGAGCTACG–3’ 5’-TAGTTTCGTGGATGCCACA-3’ 130

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MMuLV-RT.20µlofthePCRmixcontained1xbufferwithMgCl2,0.2µlofTitaniumTaqDNApolymerase,200 µM of dNTPs (Finnzymes, Finland), 250nM offorward and reverse primers (Sigma Genosys, India),and 1:50,000 diluted SYBR green dye (Amersham,India). Thermal cycling was carried out with initialdenaturation at 95˚C for 30 sec, followed by 45 cycles of denaturation at 94˚C for 30 sec, annealing at 58, 67.0, 64 and 69.0˚Crespectivelyforactin,CXCL1,TLR2,TLR4and TLR9, and extension at 72˚C for 30 sec and then the fluorescence was measured. Product specificity was confirmed by the presence of a single peak in the melting curveanalysis.ThefolddifferenceofmRNAforeachofthesechemokines,relativetothehousekeepinggenebetaactin,wascalculatedbynormalizingthethresholdcycle(Ct)valuesofchemokinesandTLRwiththatofthehousekeepinggene,actinusingtheOpticon3.1softwareprogram(Biorad,USA)ontheChromo4instrument.

Statistical analysis: Significance of differences between groupswasassessedusingonewayanalysisofvariance(ANOVA or Kruskal-Wallis test) with post-hoc tests(TukeyorDunn’s)fordifferencesbetweenindividualgroups.Two-tailedP

the enteropathogens was modulated by the presenceof commensal lactobacilli. LGG inhibited the IL-8responseofHT29cellstoV. cholerae, S. TyphimuriumandEHEC,butnot toS. flexneri.TheeffectofLGGwas more distinctive when the commensal bacteriawere present in higher numbers (10:1 compared topathogen) and with co-culture more than with pre-incubation (Fig. 1). In T84 cells, LGG significantly inhibited IL-8 secretion caused by S. TyphimuriumandS. flexneri,whenpre-incubatedwithcellsinhighconcentration(Fig.2).

Comparison of LGG and L. plantarum: LGG andL. plantarum both produced similar changes inenteropathogen-induced IL-8 secretion, and thesedifferencesbetweenthetwoLactobacillusspecieswerenot significantly different (Fig. 3). Similar results (not shown)wereobtainedusingT84cells.

Bacteria effect on gene expression of CXCL1, CCL5 and CXCL5: Significant upregulation of gene expression forCXCL1wasnotedinresponsetoS. Typhimurium,S. flexneri and EHEC (Fig. 4). Interestingly neitherofthetwostrainsofV. choleraeupregulatedCXCL1expression. The commensal bacterium LGG did notalterCXCL1expression.NoneofthepathogensalteredCCL5expressioninHT29cells(Fig.5).CXCL5wasnotdetectablebyPCRinanyofthecellcultureswithorwithoutpathogenincubation.

Gene expression of TLR 2, 4, 5 and 9 in HT29 and T84 cells: S. Typhimurium and V. cholerae O139upregulated TLR4 mRNA expression in HT29 cells(Fig.6).Theincreaseingeneexpressionwasattenuatedby simultaneous exposure to LGG. Expression ofTLR2, TLR5 and TLR9 did not significantly increase afterexposureofHT29cellstoS. Typhimurium,butV.

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Fig. 3. ComparisonofeffectofL.rhamnosusGG(LGG)andL. plantarum (Lp)oninterleukin-8secretionfromHT29cells inresponsetoenteropathogenicbacteria.HT29cellmonolayerswereexposedtopathogenandlactobacillieithersimultaneously(co-culture)for2hfollowed by 4 h culture after removal of bacteria, or first exposed to lactobacilli for 2 h and then to pathogen for 2 h followed by 4 h culture. V_O1=V. choleraeO1;V_O139=V. choleraeO139;S_tm=S. Typhimurium;Theratios1:1and10:1signifyLGG:pathogenratio.Valuesaremean+SEMofatleast3experimentswithduplicatewells.#P

choleraeO139reducedTLR5mRNAlevels(Fig.6).S. flexneri did not induce any significant change in TLR expression while EHEC reduced expression of bothTLR5andTLR9(Fig.6).

Discussion

The present studies show that several bacterialpathogens induced IL-8 secretion and activationof CXCL1 in two intestinal epithelial cell linesrepresentative of surface and crypt cell types.

LactobacilliwereshowntovariablymodulatetheIL-8 response. The epithelium is the first line of defense against entry of enteropathogenic bacteria from thelumenoftheintestineandcolon,andreleasesmediatorsaspartoftheinnatedefensesofthehost21.Atthesametimethecommensalbacteriaresidentinthecolonhavea protective effect in maintaining mucosal immunetolerance22.

Bacteriacauseintestinaldiseasethroughavarietyofdifferentmechanisms.Therationaleforthesestudieswastodirectlycomparetheabilityoforganismsusingthese different pathogenetic mechanisms to inducechemokine responses in the intestinal epithelium,using two cell lines as surrogates of the normalepithelium. While all the pathogenic bacteria testedinduced secretion of IL-8 from HT29 andT84 cells,V. cholerae induced extremely high levels of IL-8secretion fromHT29cells,butonlya tenthasmuchfromT84cells.Salmonella inducedequivalent levelsofIL-8secretionfrombothHT29andT84cells.ThenoninvasiveV.cholerainducedamuchmoremarkedinflammatory mediator response in villus-like cells thanincrypt-likecells.Ontheotherhand,theinvasiveSalmonella stimulated IL-8 secretion from crypt-likecells in equal measure as from villus-like cells.Thisprovidessupporttothesuggestionthatepithelialcellsmightrecognizepathogenicmicroorganismsvia theircapability to access defined anatomical localizations, one such restricted site being the crypt23.Vibrio andSalmonella are both flagellated organisms and thus likely to elaborate flagellin that will be recognized by theinnateimmunesystemviaTLR55,24.Inthepresentstudy, V. cholerae downregulated TLR5 expressionin epithelial cells, while upregulating TLR4. Thesignificance of this finding remains unclear. Studiesinintestinalepithelialcelllinessuggestthatprolongedexposure to lipopolysaccharide (LPS) can result intolerance to flagellin25. In monocytes and monocyte-macrophagecell lines,LPS induceda stateof cross-tolerance to flagellin by blocking downstream signalingfromTLR5.Thus,bacterialpathogenshaveevolvedavarietyofmechanismstoeludetheimmunesystemandfavour theirresidencein thehumanhost.ThepresentdemonstrationthatTLR5expressionwasdownregulatedbyV. cholerae isanotherfacetof thissurvivalabilityofpathogens.TheroleofTLR4inthechemokine response induced by V. cholerae remainstobeelucidated.EHECincreasedIL-8secretionfromtheHT29cellline.AlthoughtheeffectofEHECwasnotexaminedinT84cells,inductionofIL-8secretion

Fig. 4. Effect of pathogens on mRNA expression of CXCL1 inHT29cells.LGG=Lactobacillus GG; V_O1=V. choleraeO1;V_O139=V. choleraeO139;S_tm=S. Typhimurium; Sh_fl=Sh. flexneri;EHEC=enterohaemorrhagicEscherichia coli.Valuesaremean+SEMof3experimentsinduplicate.#P

by EHEC has been reported earlier in T84 cells26.S. flexneri, a non flagellated but invasive enteropathogen, induced IL-8 secretion in smaller amounts comparedtotheotherenteropathogens.Sincethesebacterialackflagellin, the predominant signal for IL-8 secretion comesvia lipopolysaccharide27 and this couldbe thereasonforlowerIL-8secretioncomparedtotheotherbacteria. Also, in monocyte-macrophages, Shigellahas been shown to induce apoptosis in the cells thatthen leads to reduction in pro-inflammatory cytokine production28.

In this study, gene expression of CXCL1 wasincreased in response to the invasive and adherentbacteriabutnottoV. cholerae.TheexpressionofCCL5,achemoattractantofTlymphocytes,wasnotincreasedinresponsetoanyofthebacteria.Surprisingly,expressionof epithelial neutrophil activating peptide (CXCL5)wasnotdetectedbyPCRineithercell linebeforeorafter contact with bacteria. Enteropathogen-induced

IL-8secretionfromepithelialcellswasmodulatedbyLGG and L. plantarum. These commensal probioticbacteriainhibitedtheIL-8responsetoallthepathogensstudiedwiththeexceptionofShigella.Increasingtheratio of commensal to pathogenic bacteria resultedin a numerically (not statistically) greater inhibition.Bothco-cultureandpreincubationwiththecommensalwereeffectiveininhibitingIL-8secretion.L. reuteriihasbeenreportedtoreduceIL-8secretionfromHT29andT84cellsinresponsetoS. Typhimurium29.InthisstudybothLGGandL. plantarum induced this anti-inflammatory response.

Chemokine gene expression induced byenteropathogenic bacteria in the epithelial cells wasmodulated by LGG.The modulatory effects on IL-8geneexpressionparalleledtheIL-8secretoryresponse.Attenuation of CXCL1 expression was mostly notedwith higher concentrations of Lactobacillus. As thefrontlineofthemucosalimmunesystem,theintestinal

Fig. 6. GeneexpressionoftheToll-likereceptors(TLR)2,4,5and9inHT29cellsinthebasalstateandafterexposuretopathogenicbacteria.V_O139=V. choleraeO139;S_tm=S. Typhimurium; Sh_fl=Sh. flexneri;EHEC=enterohaemorrhagicEscherichia coli.Allvaluesshownaremean+SEMof3experimentsinduplicate,normalizedtoexpressionofbeta-actin.@P

epitheliumisconstantlyexposedtolargeamountsofavarietyofTLRligands.Thepresentstudysuggeststhatenteric pathogens upregulate or downregulate specific TLRs in epithelial cells and this is an additionalmechanismthatimpactsonhostimmunityanddiseasedevelopment. These effects on TLR expression arelikelytoexplainthedifferentialeffectsofthesebacteriaonIL-8secretionbyintestinalepithelialcells.

Inconclusion,bacterialentericpathogensinducedpro-inflammatory gene expression in intestinal epithelial cell lines leading to secretion of IL-8, apotent chemoattractant of neutrophils. Differencesin their ability to elicit this responsemaydependonboth epithelial cell type and the specific pathogen. Alteration of TLR expression in epithelial cells byentericpathogensmaybeofimportanceinthegenesisof the specific chemokine response. Lactobacilli prevented the activation of pro-inflammatory genes and secretion of cytokines. Although the effects oflactobacilli indiarrhoeaaremostclearlydocumentedfor LGG and in the case of rotavirus diarrhoea, it islikely that probiotic lactobacilli will have roles inpreventingandamelioratingotherdiarrhoealillnesses.Theeffectoflactobacilliinantagonizingepithelialcellchemokineexpressionbypathogenicbacteriamaybeof relevance to their therapeutic efficacy in preventing oramelioratinginfectivediarrhoea.

Acknowledgment Authors acknowledge the Council of Scientific and Industrial Research, New Delhi, India for financial support. One of the author (PS)wassupportedbyaSeniorFellowshipfromtheIndianCouncilof Medical Research, New Delhi.The laboratory was supportedbyaFISTgrant from theDepartmentofScience&Technology,GovernmentofIndia,NewDelhi.

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