Flagellin concentrations in expectorations from cystic fibrosis patients

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Flagellin concentrations in expectorations from cystic fibrosis patients
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Balloy, Viviane
Thevenot, Guiti
Bienvenu, Thierry
Morand, Philippe
Corvol, Harriet
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Background: The aim was to measure flagellin concentrations in the expectorations of CF patients and to examine whether there are correlations with the level of respiratory insufficiency and inflammation. Methods: Sputum samples from 31 adult patients chronically colonized with P. aeruginosa were collected and analysed for their content of flagellin and IL-8. Clinical data were extracted from patient files. Results: Regardless of whether patients are colonized with mucoid strains or not, they carry clones of P. aeruginosa that express flagellin. While flagellin was present in airways of all of our CF patients, it is difficult to ascertain its contribution to inflammation (IL-8) and lung function deterioration. Conclusions: This is the first demonstration that flagellin is present in the sputum of patients. Thus, attempts to down regulate inflammation by the use of TLR5 (flagellin receptor) antagonists remain a possibility. However, this result needs to be extended to a larger number of patients to validate it for future research on this subject. Keywords: Sputum, Chronic inflammation, Infection, Respiratory function
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Balloy et al. BMC Pulmonary Medicine 2014, 14:100 http://www.biomedcentral.com/1471-2466/14/100; Pages 1-7
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doi:10.1186/1471-2466-14-100 Cite this article as: Balloy et al.: Flagellin concentrations in expectorations from cystic fibrosis patients. BMC Pulmonary Medicine 2014 14:100.

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© 2014 Balloy et al.; licensee BioMed Central Ltd. This is an Open Access article distributed under the terms of the Creative Commons Attribution License (http://creativecommons.org/licenses/by/2.0), which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly credited. The Creative Commons Public Domain Dedication waiver (http://creativecommons.org/publicdomain/zero/1.0/) applies to the data made available in this article, unless otherwise stated.
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RESEARCHARTICLEOpenAccessFlagellinconcentrationsinexpectorationsfrom cysticfibrosispatientsVivianeBalloy1,2,GuitiThvenot3,ThierryBienvenu4,5,PhilippeMorand3,6,HarrietCorvol7,8,AnnickClement7,8, ReubenRamphal9,10,DominiqueHubert3,11andMichelChignard1,2*AbstractBackground: TheaimwastomeasureflagellinconcentrationsintheexpectorationsofCFpatientsandtoexamine whethertherearecorrelationswiththelevelofrespiratoryinsufficiencyandinflammation. Methods: Sputumsamplesfrom31adultpatientschronicallycolonizedwith P.aeruginosa werecollectedand analysedfortheircontentofflagellinandIL-8.Clinicaldatawereextractedfrompatientfiles. Results: Regardlessofwhetherpatientsarecolonizedwithmucoidstrainsornot,theycarryclonesof P.aeruginosa thatexpressflagellin.WhileflagellinwaspresentinairwaysofallofourCFpatients,itisdifficulttoascertainits contributiontoinflammation(IL-8)andlungfunctiondeterioration. Conclusions: Thisisthefirstdemonstrationthatflagellinispresentinthesputumofpatients.Thus,attemptsto downregulateinflammationbytheuseofTLR5(flagellinreceptor)antagonistsremainapossibility.However,this resultneedstobeextendedtoalargernumberofpatientstovalidateitforfutureresearchonthissubject. Keywords: Sputum,Chronicinflammation,Infection,RespiratoryfunctionBackgroundCysticfibrosis(CF)ischaracterizedatthelunglevelbya floridchronicinflammatoryresponsewhichisduetomany factors,includingproteasessuchasneutrophilelastasebut alsochronicrespiratoryinfect ion,involvingmostfrequently Pseudomonasaeruginosa ( Pa )inadultpatients[1,2].Pulmonaryexacerbationsoccur,possiblyduetochangesinthe metabolismorphenotypeof Pa [3,4],andcontributetodeteriorationofthepatient ’ srespiratorystatus.Ofnote,the effectofgenderandthemenstrualcyclehasbeenshownto affect Pa phenotypes[5].Thuschroniccolonizationofthe lungswith Pa isconsideredtobetheleadingcauseofrespiratorymorbidityandmortalityinCFpatients[6,7]. Theinflammatoryresponseto Pa isbelievedtobemediatedmainlybyToll-likereceptors(TLR)4and5[8-10], andpossiblybyTLR2[11].Thusitmaybehypothesized thatbothlipopolysaccharides(LPS)andflagellinexpressed andreleasedby Pa playcrucialrolesinlunginflammation sinceTLR4and5,arebothexpressedbyalveolar macrophages,neutrophilsandrespiratoryepithelialcells [11-14].SomereportsseemtoindicatethatflagellinispossiblyanimportantagonisttoinitiateaninflammatoryreactionasLPSisbelievedtobepoorlyrecognizedbyhuman respiratoryairwaycells[15,16] .Nevertheless,inacutelung infectionsofmice,therecognitionofLPSbytheairways appearstobeeffectiveingeneratingaprotectiveinflammatoryresponse[10],thereforeonecannotexcludearolefor LPSininducingthechronicinflammatoryresponse.Indeed,proinflammatorylipidAalterationshavebeenreportedin Pa strainsofCFpatients[17]. IncontrasttolipidA,thesignalingpartoftheLPSmolecule,whoseexpressionisess entialforbacteriaviability, theexpressionofflagellinby Pa colonizingthelungsmay fluctuateandnonflagellatedstrainsarefrequentlyisolated fromCFpatients[18].Forexample, Pa growinginmucopurulenthumanrespiratorymucusfromCFpatientsrepressestheexpressionofitsf lagellin[19],inresponseto thepresenceofneutrophilelastaseinsuchmucus[20].Itis hypothesizedthatthesechang esareanattemptbythebacteriumtominimizetheinnateimmuneresponseagainstit. Furthermoreneutrophilelastaseisalsoabletodirectly cleaveflagellin,resultinginthelossofitsabilitytoinduce *Correspondence: chignard@pasteur.fr1UnitdeDfenseinneetInflammation,InstitutPasteur,Paris,France2DfenseinneetInflammation,InsermU874,InstitutPasteur,25,rueduDr Roux,Paris75015,France Fulllistofauthorinformationisavailableattheendofthearticle 2014Balloyetal.;licenseeBioMedCentralLtd.ThisisanOpenAccessarticledistributedunderthetermsoftheCreative CommonsAttributionLicense(http://creativecommons.org/licenses/by/2.0),whichpermitsunrestricteduse,distribution,and reproductioninanymedium,providedtheoriginalworkisproperlycredited.TheCreativeCommonsPublicDomain Dedicationwaiver(http://creativecommons.org/publicdomain/zero/1.0/)appliestothedatamadeavailableinthisarticle, unlessotherwisestated.Balloy etal.BMCPulmonaryMedicine 2014, 14 :100 http://www.biomedcentral.com/1471-2466/14/100

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aninnatehostresponse[21].Inaddition,itisthoughtthat Pa withinamicrocolonyorbiofilm-growingmucoid(alginate-producingstrains)possessauniquephenotypequite differentfromtheirplanktonic counterparts[22,23],repressinggenesforflagellarbiogenesisinadditiontoothers [24,25].Henceonecaneasilyconcludethatflagellinmayor maynotbeexpresseddependingontheenvironment,and thusmayormaynotplayaroleinthechronicinflammatoryresponse. TheaimofthepresentstudywasthustomeasureflagellinconcentrationsintheexpectorationsofCFpatientsand toexaminewhethercorrelatio nsexistwiththepresenceor absenceofflagellinandthelevelofrespiratoryinsufficiency. Thisisanimportantquestionasincaseofthepresence andofapositivecorrelationonecanenvisageantiinflammatorytreatmentbasedontheinhibitionofthe interactionbetweenflagellinandTLR5bymutantsofflagellinoranti-TLR5smallmolecules.Indeed,ithasbeen alreadyshownwithhumanrespi ratoryepithelialcellsthata mutantofflagellinthatactivatesTLR5poorly,wasableto reduceIL-8synthesistrigger edbywild-typeflagellin[26].MethodsPatientsThirty-oneadultswithCFattendingtheCFcentreat CochinUniversityHospital,Pariswereenrolledbetween May2011andOctober2011duringaplannedvisit.None ofthepatientswashospitalized .Criteriaforinclusionwere: ageolderthan18andchroniccolonizationwith Pa (definedaccordingtotheLeecriteria[27]whenairwaysampleswere Pa culturepositivein>50%oftheexplored monthsoverthelast12month s).Clinicaldatawereextractedfrompatientfiles,includingdemographicdata(age andgender),CFTRgenotype,exocrinepancreaticinsufficiency,diabetes,bodymassindexandpulmonaryfunction. Pulmonaryfunctiontestswereperformedonthedayofthe outpatientvisit.Forcedexpiratoryvolumeinonesecond (FEV1)andforcedvitalcapacity(FVC)wereexpressedas percentagesofthepredictedvalue(%pred.).Patientswere consideredstableorinexacerbationaccordingtoFuchscriteria[28].Theygavetheirinformedconsentforparticipationinthestudywhichwasconductedinaccordancewith theDeclarationofHelsinkiandFrenchlawandwasapprovedbytheInstitutionalReviewBoardforMedicalResearch(CCTIRS#08 – 370).Inaddition,patientsprovided consentforthepublicationoftheirdata.SputumcollectionandtreatmentAllsputawerespontaneouslyexpectoratedandwerecollectedinasterilecontainer.Afractionoftheeachsputum samplewasprocessedbytheClinicalMicrobiologyLaboratoryofthehospitalformicrobiologicalanalysis. Pa was quantifiedbyplattingserialdilutions(1/2,1E-3,1E-5)of fluidified(Digest ’ Eur,Eurobio,Courtaboeuf,France)sample onnon-selective(COHandPVX,BioMerieux,Marcy L ’ Etoile,France)andselective(Dirgalski,BioRad,Marnes LaCoquette,France)solidmedia.Plateswereincubatedfor upto7days.Quantificationandmucoidphenotype(absence/presenceofviscousandslimycolonies)ofeach Pa isolatewasreporteduponbacterialisolation.Theother fractionofthesputumwasleftoniceandprocessedwithin twohoursfromthestartofthecollection.Allmaterialwas transferredtoaPetridishtod iscardsaliva,thenthesputum wascollectedandvortexedfor1minwithv/vRIPAlysis buffer2(300mMNaCl,50mMHEPES,10mMEDTA, 0.2%SDS,2%NonidetP40,0.1%Deoxycholate),supplementedwithComplete,EDTAfree-proteaseinhibitorcocktail(RocheDiagnostics).Sampleswerecentrifugedandthe supernatantwasstoredat 80C.FlagellinquantificationFlagellinamountsintheexpectorationswerequantifiedby WesternBlotanalysisbycompa ringthebandintensitytoa standardcurveofpurifiedPseudomonasflagellin,purified asdescribedpreviously[26]. Forthequantification,totalp roteinconcentrationsinexpectorationsweremeasuredusingPierceBCAprotein assayandthensolubilizedwithLaemmlibufferpriorto electrophoresis.Fourconcentr ationsofprotein(1,2.5,5 and10 g/well)fromoneexpectorationandthestandard curveofflagellin(0.1,0.25,0.5,and1.0ng/well)were loadedona10%acrylamidegelandfractionatedbySDSPAGE.Proteinswereelectrotransferredtoapolyvinylidene difluoridemembrane(Immobilon,MilliporeCorp.,Bedford, MA)andprobedusingspecificantibodyagainstPseudomonasflagellinwhichrecognizesthetwodifferenttypes, flagellinsaandbof Pa whichsharelargestretchesamino acidsequences[29].Boundantibodywasdetectedusing theECL+immunoblottingdetectionsystem(Thermofisher, Rockford,USA)accordingtothemanufacturer ’ sinstructions.Molecularmasseswereestimatedfromcalibration standardsincludedineachgel. BandintensitywasanalyzedbyusingImageJsoftware version1.45g.Astandardcurveofpurifiedflagellinwas constructedusingalinearcurve-fitbyplottingtheband intensityforeachstandardonthey-axisagainsttheconcentrationonthex-axis.Flagellinconcentrationsinexpectorationswerecalculatedusingtheequationofthe standardcurve.Thiscalculationwasmadeforeachofthe threeorfourconcentrationsofproteins,andthevalues wereaveraged.Tonormalizere sults,flagellinconcentrationswereexpressedfor100 goftotalproteins.RepresentativeimmunoblotsareshowninFigure1.Interleukin-8determinationIL-8concentrationsinexpectorationsweredetermined usingDuo-SetELISAkit(R&Dsystems,Minneapolis, USA).Balloy etal.BMCPulmonaryMedicine 2014, 14 :100 Page2of7 http://www.biomedcentral.com/1471-2466/14/100

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StatisticalanalysisTheanalysisofvariance(ANOVA)wasperformedformultiplecomparisonswithBonferroniposttest.Forcomparison oftwosamplesStudent ’ s t testwasapplied.Apvalue<0.05 wasconsideredsignificant.GraphPadPrism5(GraphPad SoftwareInc,SanDiego,CA)wasusedfortheanalysis.ResultsPatientdemographics,clinicalstatusandflagellin concentrationsTheclinicalcharacteristicsofthepatientsandresultsof flagellinconcentrationsinsputumareshowninTable1. PatientswereclassifiedintwogroupsofrespiratoryinsufficiencyaccordingtotheirFEV1:severerespiratory insufficiencybelow45%pred.andmoderaterespiratory insufficiencyabove45%pred. Themeanageofthepatientswithsevererespiratoryinsufficiencywas31.89.2years,itwasof31.56.6forthose withmoderaterespiratoryins ufficiency(ns).Wedidnot observeaclear-cutdifferenceinthe CFTR genotypebetweenthesetwogroupsofpatients(8/17ofthepatients withseveredisease(47%)wereF508delhomozygotescomparedto6/14ofthepatientswithmoderatedisease(43%)). Comparisonsofthetwogroupsforthedifferentclinicalparameterssuchaspancreaticinsufficiency(PI) vs pancreaticsufficiency(PS),cysticfibrosis-relateddiabetes(CFRD)andbodymassindex(BMI)didnotdemonstrateobviousdifferencesaswell(Table1). IL-8measurements,doneasaproxyfortheongoing inflammatoryresponsealsodidnotdemonstratea differenceaccordingtothecategoryofrespiratoryinsufficiencyofourpatients(Table1).Theconcentrations rangedfrom966to10,848ng/100 gproteins. Finally,fortheparameterconstitutingtheaimofour study, i.e. theconcentrationofflage llininthesputum,the differencewasnotstatistica llysignificantbetweenthetwo groups,withmeanvaluessdof14.015.6ng/100 gproteinsforpatientswithsevererespiratoryinsufficiencyand of9.110.1ng/100 gproteins(Table1).Correlationofflagellinconcentrationsinsputumwith respiratoryinsufficiencyInordertovisualizedifferentlyapossiblecorrelationbetweenflagellinconcentrationsa ndpatientrespiratoryinsufficiency,weconsideredtwogroups, i.e. agroupwith flagellinconcentrationslowerthan10ng/100 gproteins (20patients),andagroupwithflagellinconcentrations higherthan10ng/100 gproteins(11patients).The meansdvaluesforflagellinwerethus4.22.0and 25.514.7ng/100 gproteins,respectively(Table2). Forthegroupofpatientswithlowflagellinconcentrationsthemeanagewas31.16.8years,witha50/50 male/femaleratio,andforthegroupofpatientswith highflagellinconcentrationsthemeanagewas32.6 10.1years,with54/46male/femaleratio.Meanssd valueswerenotstatisticallysignificant. InfactforFEV1therewasnosignificantdifferencesas well,valuesbeingof52.118.1%ofpredictedand42.0 17.5%(meanssd)ofpredictedforthelowandhigh flagellinconcentrationgroups,respectively.Thus,higher flagellinconcentrationsdidnotautomaticallysuggest moreseveredisease. Wealsoassessedwhetherhighconcentrationsofflagellin maybelinkedwithpulmonaryexacerbations i.e. acute worseningofCFsymptomscausedbyinfectionandthat leadtotheneedforadditionalan tibiotictreatment[7].Five patientshadmildpulmonarye xacerbationrequiringan antibioticIVcourseathome,nonewashospitalized.No correlationwasobservedaspatientswithexacerbationhad verydifferentconcentrationsof flagellinintheirexpectoration,rangingfrom0.64to60.65ng/100 gproteins,infact thetwoextremevaluesofthewholegroupof31patients. Amongthe20patientswithflagellinconcentrationslower than10ng/100 gproteins,4patientshadexacerbations, comparedtoonlyonepatientamongthe11otherpatients whoseflagellinconcentrationswerehigher.Thus,there wasnocorrelationbetweenfl agellinconcentrationsand exacerbations. Analysisoftheexpectorationsdidnotdemonstrate differencesamongthetotalproteinsinsputumbetween thetwogroups(Table2).Aswell,nocorrelation(r2= 0.02)couldbefoundbetweenflagellinconcentrations andbacterialnumbers. Figure1 Representativewesternblotsofthreesputumextracts containinglow(n36),medium(n25)andhigh(n9)flagellin concentrations. Fourconcentrationsofprotein(1,2.5,5and10 g/ well)from3differentexpectorationsandstandardcurveofflagellin (0.1,0.25,0.5,and1.0ng/well)wereloadedon10%acrylamidegel andfractionatedbySDS-PAGE(seeMethods).Notei)thatthe recombinantnonglycosylatedflagellin(righthandside)hasa molecularweightlower(40kD)thanthenativeflagellin(lefthand side)presentinthesputum(49kD)andcorrespondingtoflagellin typeb,and2)thepresenceafaintbandwitha40kDmwobserved forpatients25and9,andcorrespondingtoflagellintypea. Balloy etal.BMCPulmonaryMedicine 2014, 14 :100 Page3of7 http://www.biomedcentral.com/1471-2466/14/100

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Interestingly,mucoidstrainswerepresentin4/20 patients(20%)withlowflagellinconcentrationsand evenmorein4/11(36%)patientswithhighflagellin concentrations.Asdiscussedpreviouslythegeneralstatementismadethatmucoidstrainslosetheabilityto makeflagellin,howeverregardlessofwhetherthepatient Table1Patientswithsevere vs moderateclinicalseverityNSexFEV1FVCPI/PSCFRDBMIIL-8Flagellin (%pred.)(%pred.)kg/m2(ng/100 gprot)(ng/100 gprotein) Patientswithsevererespiratoryinsufficiency 2F3038PIN15.042189.7211.23 3M2455PIY24.773163.505.76 8M4443PIN20.013064.522.92 9M2039PIY20.681866.8623.29 10M4062PIY16.614009.7433.78 13M4460PIN19.355851.318.58 14M3769PSN20.43966.3631.53 15M3046PIN18.947007.4813.40 16F2737PIN21.2210848.5360.65 17M2957PIN22.402754.3016.75 20M4358PIN21.262203.664.24 21F4358PIN20.503596.656.50 25F4260PIY33.204089.033.68 31M3858PIN21.412681.307.17 34F1822PIY21.683050.021.74 35F4154PSN19.201735.266.12 36F4161PIN18.263764.780.64 MeanM:58.8%34.7651.59PI:88.2%CFRD:29.4%20.883696.6514.00 SD8.7812.113.872361.0215.60 Patientswithmoderaterespiratoryinsufficiency 4F6681PIN19.891114.5312.66 5F6679PIY25.331680.154.80 6F6473PSN26.672748.145.10 7M6068PIN22.592548.475.62 11F91106PIN21.641832.333.41 12M5979PIY25.697971.282.68 18F7084PIN25.812289.973.62 19F7382PIY17.752142.7230.01 22M75108PIN19.594286.103.82 23F5589PIY20.261648.2913.88 24M4674PIY20.863194.504.50 26M5555PIY23.663217.8532.80 37F5782PIY23.314453.101.07 38M7691PIN22.993437.082.73 MeanM:42.8%65.2182.21PI:92.9%CFRD:50%22.573040.329.05 SD11.3513.802.701725.9710.15Comparisonoftwogroupsofpatients,agroupwithclinicalseveritydefinedassevere(18patients),andagroupwithclinicalseveritydefinedasmod erate (13patients). FEV1(%pred.)andFCV(%pred.)areforcedexpiratoryvolumeinonesecondandforcedvitalcapacity(expressedaspercentageofthepredictedvalue), respectively.Pancreaticinsufficiency(PI) vs pancreaticsufficiency(PS);cysticfibrosis-relateddiabetes(CFRD);Bodymassindex(BMI).Balloy etal.BMCPulmonaryMedicine 2014, 14 :100 Page4of7 http://www.biomedcentral.com/1471-2466/14/100

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Table2Patientswithhigh vs lowflagellinconcentrationsNSexStatusMucoid/non mucoid FEV1FVCTotalproteinsIL-8Flagellin Stable/Exacerbation(%pred.)(%pred.)(mg/ml)(ng/100 gprot)(ng/100 gprotein) Patientswithflagellinsputum<10ng/100 gprotein 3MStableNonmucoid24556.583163.505.76 5FStableNonmucoid66793.211680.154.80 6FExacerbationMucoid64734.572748.145.10 7MStableNonmucoid60689.342548.475.62 8MExacerbationNonmucoid44433.843064.522.92 11FStableNonmucoid911063.151832.333.41 12MStableNonmucoid59794.397971.282.68 13MStableNonmucoid446011.85851.318.58 18FStableNonmucoid70844.172289.973.62 20MStableNonmucoid43588.682203.664.24 21FStableMucoid43585.913596.656.50 22MStableNonmucoid751085.874286.103.82 24MStableNonmucoid46747.253194.504.50 25FStableNonmucoid42604.914089.033.68 31MStableMucoid38584.702681.307.17 34FExacerbationNonmucoid18224.223050.021.74 35FStableNonmucoid41547.831735.266.12 36FExacerbationNonmucoid41616.713764.780.64 37FStableMucoid57828.934453.101.07 38MStableNonmucoid76916.593437.082.73 MeanM:50%non.mucoid:.80%52.1068.656.133382.064.24 SD 18.1120.392.301491.932.03 Patientswithflagellinsputum>10ng/100 gprotein 2FStableMucoid30387.902189.7211.23 4FStableNonmucoid66813.251114.5312.66 9MStableMucoid20398.221866.8623.29 10MStableMucoid40624.114009.7433.78 14MStableNonmucoid37695.38966.3631.53 15MStableNonmucoid30466.427007.4813.40 16FExacerbationNonmucoid27375.8710848.5360.65 17MStableNonmucoid29572.792754.3016.75 19FStableNonmucoid73825.142142.7230.01 23FStableMucoid55896.811648.2913.88 26MStableNonmucoid55558.443217.8532.80 MeanM:54.5%Nonmucoid:.63.6%42.0059.55 5.85 3433.3125.45 SD 17.5218.801.942981.8614.65Comparisonoftwogroupsofpatients,agroupwithflagellinconcentrationslowerthan10ng/100 gproteins(20patients),andagroupwithflagellinconcentrationshigherthan10ng/100 gproteins(11patients). FEV1(%pred.)andFCV(%pred.)areforcedexpiratoryvolumeinonesecondandforcedvitalcapacity(expressedaspercentageofthepredictedvalue), respectively.Balloy etal.BMCPulmonaryMedicine 2014, 14 :100 Page5of7 http://www.biomedcentral.com/1471-2466/14/100

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carriedornotamucoidstrainof Pa ,themeanconcentrationofflagellinfoundinthesputumsampleswas similar, i.e. 13.611.4 vs 12.515.2ng/100 g,respectively.Itshouldhoweverbenotedthattheclinicallaboratoryreportedthepresenceornotofamucoidstrain whichdoesnotimplythatwhenthisphenotypewas present,thenonmucoidstrainwasabsent.DiscussionFlagellinisapotentactivatorofabroadrangeofcell typesinvolvedininnateandadaptiveimmunity.ItsrecognitionbyTLR5isinvolvedinactivatingpulmonary defensesagainst Pa thatleadseventuallytoelimination ofthisbacteriumfromthehost.Thus,bacterialmutants thatlackedflagellinappearedtoevadeimmunecontrol andwererelativelyslowlyclearedfromthelungs.Along thesameline,thelossoftheTLR5responsehasconsequencesonthehostresponse,thatresultsinanimpairmentofantimicrobialeffectors[30]. ThewellknownparadoxisthattheTLR5-flagellin interactionisamajormediatorofinflammationfollowingexposureto Pa .Indeed, Pa mutantswhichoverproducedflagellin,causedsevereinflammation[31].The consequenceisthatsomeauthorshaveproposedTLR5 asananti-inflammatorytarget[32].Interestingly,CFpatientscarryingtheTLR5prematurestopcodonhada higherbodymassindexthanCFpatientshomozygous forthefunctionalallele.Thisissomeevidencethata lossofTLR5functionresultinginreducedflagellinresponsivenessisassociatedwithimprovedhealthindicatorsinadultswithCF.Unfortunately,improvementsin lungfunctionswerenotstatisticallysignificant[32].Anotherstudydemonstratesthatflagellininducesthegenerationofmyeloid-derivedsuppressorcellsandsuggest that Pa usesthismechanismtoundermineTcell – mediatedhostdefenseinCF[33].Insummary,thereisnota clearpictureaboutthebeneficialordeleteriousconsequencesofTLR5-flagellininteraction invivo .Andin fact,itwasevennotknownwhetherflagellinwas presentornotinthesputaofCFpatients.ConclusionThepresentstudyalthoughhavingasmallsamplesize,allowstwoconclusions.Firstly,itpointsoutthatpossiblyall Pa colonizedpatients,regardle ssofwhethertheclinicallaboratoryindicatestheyarecolonizedwithmucoidstrains, carryclonesof Pa thatexpressflagellin.Thisisthefirst timethatflagellinconcentrationsinthesputumofCFpatientshasbeenexamined.Thuscautionshouldbeexercised inconcludingthatpatientswithmucoidstrainsceaseto carryflagellinproducingclone s.Thisfindingisconsistent witharecentreportthatthereissignificant “ phenotypic ” heterogeneityof Pa populationsinaCFpatient[34].Secondly,whileflagellinseemstobealwayspresentinCF airways,itisdifficulttomeasur eitscontributiontoinflammation(IL-8measurements)andlungfunctiondeterioration,sincewecouldnotcorrelateamountspresentwith thelevelofrespiratoryinsufficiencynorwiththepresence ofapulmonaryexacerbation.Howevergiventhatthisagonisthasnowbeenshowntobepresentinallpatientswe studied,attemptstodownregulateinflammationbytheuse ofTLR5antagonistsstillremainviable. Althoughweareawarethatthepresentdataarenot conclusiveandneedtobestrenghtened,webelievethat theirnovelty(thisisthefirstdemonstrationthatflagellin ispresentinthesputumofallpatientsstudied)deserves tobebroughttotheattentionoftheCFcommunity.Competinginterests Theauthorsdeclarethattheyhavenocompetinginterests. Authors ’ contributions VBcarriedoutthelaboratoryexperiments(proteinmeasurements,western blots,immunoassays)andperformedthestatisticalanalysis.GTcarriedout thelaboratoryexperiments(preparationoftheexpectorations).TBanalyzed andprovidedpatientgenotypes.PManalyzedandprovidedpatient microbiologicaldata.HCandACparticipatedinthestudydesign.RR participatedinthedesignofthemanuscriptandhelpedtodraftit.VB,DH andMCconceivedofthestudy,andparticipatedinitsdesignand coordinationandhelpedtodraftthemanuscript.Allauthorsreadand approvedthefinalmanuscript. Acknowledgements Wethankthenon-profitassociationVaincrelaMucoviscidoseforafinancial support. Authordetails1UnitdeDfenseinneetInflammation,InstitutPasteur,Paris,France.2DfenseinneetInflammation,InsermU874,InstitutPasteur,25,rueduDr Roux,Paris75015,France.3UniversitParisDescartes,SorbonneParisCit, FacultdeMdecine,UPRESEA2511,Paris,France.4GroupeHospitalier Cochin – Broca – HtelDieu,AP-HP,LaboratoiredeBiochimieetGntique Molculaire,Paris,France.5UniversitParisDescartes,SorbonneParisCit, InsermU1016,CNRS(UMR8104),Paris,France.6HpitalCochin,AP-HP, ServicedeBactriologie,Paris,France.7HpitalArmandTrousseau,AP-HP, PneumologiePdiatrique,Paris,France.8UPMC,UniversitParis6,Inserm, UMR-SU938,Paris,France.9UniversitFranoisRabelais,UMR1100,Tours, France.10INSERM,UMR1100/EA6305,Tours,France.11HpitalCochin,AP-HP, ServicedePneumologie,CRCMadultes,Paris,France. Received:30December2013Accepted:13May2014 Published:9June2014 References1.CallaghanM,McCleanS: Bacterialhostinteractionsincysticfibrosis. 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PLoSOne 2013, 8: e60225.doi:10.1186/1471-2466-14-100 Citethisarticleas: Balloy etal. : Flagellinconcentrationsin expectorationsfromcysticfibrosispatients. BMCPulmonaryMedicine 2014 14 :100. Submit your next manuscript to BioMed Central and take full advantage of: € Convenient online submission € Thorough peer review € No space constraints or color “gure charges € Immediate publication on acceptance € Inclusion in PubMed, CAS, Scopus and Google Scholar € Research which is freely available for redistribution Submit your manuscript at www.biomedcentral.com/submit Balloy etal.BMCPulmonaryMedicine 2014, 14 :100 Page7of7 http://www.biomedcentral.com/1471-2466/14/100