Anti-microbial immunity is impaired in COPD patients with ... · an upper respiratory tract infection or an increase in any of the symptoms of dyspnoea, cough, sputum volume or purulence

1

Anti-microbialimmunityisimpairedinCOPDpatientswithfrequentexacerbations

AranSinganayagam1,Su-LingLoo2,MariaCalderazzo1,LydiaJFinney1,Maria-BelenTrujillo

Torralbo1,EteriBakhsoliani1, JasonGirkin2,PunnamVeerati2,PrabuddhaSPathinayake2,

Kristy S Nichol2, Andrew Reid2, Joseph Foottit1, Sebastian L Johnston1*, Nathan W

Bartlett2*,PatrickMallia1*

*Theseauthorscontributedequallytothiswork

Affiliations

1. NationalHeartandLungInstitute.ImperialCollegeLondon.UK.

2. FacultyofHealthandMedicineandPriorityResearchCentreforHealthyLungs.

HunterMedicalResearchInstituteandUniversityofNewcastle.Newcastle.NSW

2305.Australia

Correspondingauthor:DrAranSinganayagam.NationalHeartandLungInstitute.Imperial

CollegeLondon.

Keywords: chronicobstructivepulmonarydisease,exacerbation,viral infection,bacterial

infection

Wordcount:2493Conflictsofinterest:S.L.J. has personally received consultancy fees from Myelo Therapeutics GmbH, ConcertPharmaceuticals,Bayer,andSanofiPasteura,andAviragen;heandhisinstitutionreceivedconsultancyfeesfromSynairgen,Novarits,BoehringerIngelheim,Chiesi,GlaxoSmithKline,andCentocor.S.L.J.isan inventoronpatentsontheuseof inhaled interferons for treatmentofexacerbationsofairwaydiseases.Theremainingauthorsdeclarenocompetinginterestsrelevanttothismanuscript.

certified by peer review) is the author/funder. All rights reserved. No reuse allowed without permission. The copyright holder for this preprint (which was notthis version posted May 9, 2019. . https://doi.org/10.1101/632372doi: bioRxiv preprint

https://doi.org/10.1101/632372

2

ABBREVIATIONSBECs:bronchialepithelialcellsALI:airliquidinterfaceCOPD:ChronicobstructivepulmonarydiseaseFEV1:Forcedexpiratoryvolumein1secondFVC:ForcedvitalcapacityGOLD:GlobalInitiativeforObstructivelungdiseaseIFN:InterferonISG:Interferon-stimulatedgeneMBL:Mannose-bindinglectinPEFR:peakexpiratoryflowrateRV:rhinovirusSLPI:Secretoryleucocyteproteaseinhibitor


https://doi.org/10.1101/632372

3

ABSTRACT

Background:Patientswithfrequentexacerbationsrepresentachronicobstructivepulmonarydisease

(COPD)sub-group requiringbetter treatmentoptions.Theaimof this studywas todetermine the

innateimmunemechanismsthatunderliesusceptibilitytofrequentexacerbationsinCOPD.

Methods:Wemeasuredsputumexpressionofimmunemediatorsandbacterialloadsinsamplesfrom

patients with COPD at stable state and during virus-associated exacerbations. Ex vivo immune

responses to rhinovirus infection in differentiated bronchial epithelial cells (BECs) sampled from

patientswithCOPDwereadditionallyevaluated.Patientswerestratifiedasfrequentexacerbators(>2

exacerbationsintheprecedingyear)orinfrequentexacerbators(<2exacerbationsinthepreceding

year)withcomparisonsmadebetweenthesegroups.

Results:FrequentexacerbatorshadreducedsputumcellmRNAexpressionoftheanti-viralimmune

mediatorstype Iand III interferonsandreduced interferon-stimulatedgene(ISG)expressionwhen

clinicallystableandduringvirus-associatedexacerbation.RV-inductionofinterferonandISGsexvivo

wasalsoimpairedindifferentiatedBECsfromfrequentexacerbators.Frequentexacerbatorsalsohad

reduced sputum levels of the anti-microbial peptide mannose-binding lectin (MBL)-2 with an

associated increase in sputum bacterial loads at 2 weeks following virus-associated exacerbation

onset.MBL-2levelscorrelatednegativelywithbacterialloadsduringexacerbation.

Conclusion: These data implicate deficient airway innate immunity in the increasedpropensity to

exacerbationsobservedinsomepatientswithCOPD.Therapeuticapproachestoboost innateanti-

microbial immunity in the lung could be a viable strategy for prevention/treatment of frequent

exacerbations.


https://doi.org/10.1101/632372

4

INTRODUCTION

Chronic obstructive pulmonary disease (COPD) is an inflammatory airway disorder inwhich acute

exacerbations represent a major complication. Acute exacerbations are a substantial cause of

morbidityandmortality1-3andpreventingexacerbationsremainsamajorunmetneed.Therehasbeen

increasing interest in a recently identified sub-group of patients with COPD who are at risk of

exacerbations, defined as the ‘frequent exacerbator phenotype’.4 Patients with frequent

exacerbationshaveworseclinicaloutcomesincludingincreasedmorbidity1,acceleratedlungfunction

decline2andgreatermortality5,suggestingthatthisgrouprequiresspecialconsideration.Todate,the

underlyingmechanismsthatpredisposesuchpatientstomorefrequentexacerbationshavenotbeen

elucidated.Itisplausiblethatabnormalitiesinhostimmunitycouldunderlieanincreasedpropensity

toexacerbations.

Virusesareamajoraetiologicaltriggerforexacerbations6,7anddataexiststosuggestthatCOPDmay

be associated with deficient anti-viral immunity 8,9. However, not all studies have shown this

abnormality10,11,suggestingthatitmayvaryaccordingtodiseasephenotype.Frequentexacerbators

could represent one sub-group in whom defective anti-viral immunity is more prominent. Both

experimentalandnaturally-occurringexacerbationstudiesalsoconfirmthataninitialvirusinfection

canprecipitatesecondarybacterialinfectioninCOPD12,13withneutrophilelastase-inducedcleavage

ofanti-microbialpeptidesbelievedtobeimportantmechanistically12.Althoughbacterialcolonisation

at stable state has been shown to be associated with increased exacerbation frequency 14, anti-

microbial peptide responses andpropensity todevelop secondarybacterial infection during virus-

infectionsinfrequentversusinfrequentexacerbatorshasnotpreviouslybeenstudied.

WehypothesisedthatpatientswithCOPDwhohaveahistoryoffrequentexacerbationswouldhave

impaired anti-viral and/or anti-bacterial immunity compared to infrequent exacerbators. Using

analysisofsputumsamplesfromacohortofpatientsmonitoredprospectivelyinthecommunity,in

combinationwithinvitroexperimentsinBECssampledfrompatientswithCOPDanddifferentiatedat

theair-liquidinterface(ALI),weshowthatlunginnateanti-microbialimmunityisimpairedinfrequent

exacerbators.Thesemechanismsmayunderlietheincreasedpropensitytoexacerbationobservedin

somepatients.


https://doi.org/10.1101/632372

5

METHODS

TheStMary’sHospitalnaturally-occurringCOPDexacerbationcohort

Acohortof40COPDsubjectswasrecruitedtoalongitudinalstudycarriedoutatStMary’sHospital,

London UK between June 2011 to December 2013 investigating the pathogenesis of naturally-

occurringCOPDexacerbations.ThestudyprotocolwasapprovedbytheEastLondonResearchEthics

Committee(studynumber11/LO/0229)andallsubjectsgaveinformedwrittenconsent.Thesubjects

allhadaclinicaldiagnosisofCOPDthatwasconfirmedwithspirometry.Allsubjectshadaninitialvisit

atbaselinewhenclinicallystableforclinicalassessment,spirometry(forcedexpiratoryvolumein1

second(FEV1),forcedvitalcapacity(FVC)andpeakexpiratoryflow(PEF))andclinicalsamplecollection

includingspontaneousor inducedsputum,takenandprocessedaspreviouslydescribed8,15.Atthe

baselinevisit,allsubjectswereaskedaboutthenumberofexacerbationsexperiencedintheprevious

yearandweregroupedintotwogroups–frequentexacerbators(≥2exacerbationsinprecedingyear)

and infrequent exacerbators (0 – 1 exacerbations in theprevious year), as previously described16.

Subjectsthenhadrepeatvisitsatthreemonthlyintervalswhenclinicallystableandwerefollowedup

foraminimumof6months.Subjectsreportedtothestudyteamwhentheydevelopedsymptomsof

anupperrespiratorytractinfectionoranincreaseinanyofthesymptomsofdyspnoea,cough,sputum

volumeorpurulence.ExacerbationwasdefinedusingtheEastLondoncohortcriteria.2Subjectswere

seenwithin48hoursofonsetoftheirsymptomsforcollectionofsamplesandrepeatvisitswascarried

outa twoweeksafter the initialexacerbationvisit.Samples fromthiscohorthavebeenused ina

previousstudyinvestigatingtheroleofairwayglucoseinCOPD17

Experimentalchallengestudies

Forbaselineanalysesofsputumsolublemediators,inadditiontosamplestakenfromthenaturally

occurring exacerbation study, we also included baseline (pre-infection) samples from 14 COPD

subjects recruited to experimental challenge COPD studies8,18. All subjects in these studies gave

informedwritten consent and theprotocolwas approvedby StMary'sNHSTrustResearchEthics

Committee (studynumbers00/BA/459Eand07/H0712/138).Thesesubjectswerecharacterisedas

frequentorinfrequentexacerbatorsusingthesamedefinitionasdescribedabove.Sputumsamples

were processed using exactly the same methodology as described in the naturally occurring

exacerbationstudy.


https://doi.org/10.1101/632372

6

RVinfectionofair-liquidinterfacedifferentiatedbronchialepithelialcellsfrompatientswithCOPD

PrimaryBECsobtainedbronchoscopicallyfrompatientswithCOPD(GOLDStageIIorIII)weregrown

untilconfluentanddifferentiatedatairliquid-interface(ALI),aspreviouslydescribed19-21.Allsubjects

includedgave informedwrittenconsentand thestudyprotocolwasapprovedby theHunterNew

EnglandHumanResearchEthicsCommittee(05/08/10/3.09).Primarycellsweregrownincomplete

bronchialepithelialcellgrowthmedium(BEGM)(Lonza)withgrowthfactorsupplementsinsubmerged

monolayercultureandthenseededat2x105cells/mLintranswellsuntilconfluent.RV-A1infection

(MOI0.1)wasappliedtotheapicalsurfacefor2hours in250μlbronchialepithelialbasalmedium

(BEBM)minimalat35oC.Followingthis,infectionmediawasreplacedwith500μlfreshBEBMminimal

fortheremainderoftheexperiment.Sampleswerecollectedat72hourspost-infectionwithapical

mediaremovedandstoredforproteinexpressionanalyses.TypeIIIIFNproteinsweremeasuredusing

acustom-designedLEGENDplexkit(BioLegend).IFN-βwasmeasuredusingtheVerikineELISA(PBL).

HalfofthetranswellmembranewasalsocarefullycutfromtheinsertsandcollectedintoRLTbuffer

(Qiagen) containing 1% 2-mercaptoethanol for downstream molecular analyses by RT-qPCR. The

remaining transwell membrane was fixed in 10% neutral-buffered formalin for 24 hours for

histologicalcross-sectionstoconfirmdifferentiation.

Measurementofsputumproteins

Proteinlevelsincell-freesputumsamplesofCXCL10/IP-10andCCL5/RANTESweremeasuredusing

duosetELISAkits(R&DSystems),accordingtomanufacturer’sinstructions.Proteinlevelsofallanti-

microbialpeptideswereassayedusingcommercialELISAassaykits,aspreviouslydescribed12,22.The

sourcesoftheindividualELISAswereasfollows:Elafin(R&DSystems),Mannosebindinglectin(MBL)-

2(R&DSystems)andSLPI(R&DSystems).

RNAextraction,cDNAsynthesisandquantitativePCR

TotalRNAwasextracted from sputumcell pelletsorbronchial epithelial cell lysates stored inRLT

buffer(RNeasykit,Qiagen)and2µgwasusedforcDNAsynthesis(OmniscriptRTkit).QuantitativePCR

wascarriedoutusingpreviouslydescribedspecificprimersandprobesforeachgeneofinterest23,

and normalized to 18S rRNA. Reactions were analysed using ABI 7500 Fast Realtime PCR system

(AppliedBiosystems,Carlsbad,CA).


https://doi.org/10.1101/632372

7

Virusdetection,DNAextractionandbacterial16SquantitativePCR

DNAextractionfromtotalsputumwasperformedusingtheFastDNASpinKitforSoil(MPBiomedicals,

SantaAna,USA),aspermanufacturerinstructions.Bead-beatingwasperformedfortwocyclesof30

seconds at 6800 rpm (Precellys, Bertin Technologies, Montigny-le-Bretonneux, France). Total 16S

bacterial loads were measured using quantitative PCR, as previously described 24. Viruses were

detectedasdescribedpreviously12.

StatisticalAnalyses

Comparisons of sputum mRNA expression and protein concentrations between frequent and

infrequent exacerbatorswere analysed using theMann-WhitneyU test. For in vitro experiments,

baselineversusRV-inducedexpressionwasanalysedusingWilcoxonrank-sumtest.MannWhitneyU

test was used to compare RV induction of mRNA or proteins between frequent and infrequent

exacerbators. Correlations between datasets were examined using Spearman's rank correlation

coefficient. All statistics were performed using GraphPad Prism 6 software. Differences were

consideredsignificantwhenP<0.05.


https://doi.org/10.1101/632372

8

RESULTS

Studypopulation

We utilised a community-based cohort of COPD patients to evaluate anti-microbial immunity at

baseline(stable-state)andduringvirusassociatedexacerbation.Forbaselineanalysesofsputumcell

mRNA(interferons(IFNs)andinterferon-stimualatedgenes(ISGs)),36patientshadsufficientsample

for evaluationand,of these, 13patients (36.1%)were classified as frequentexacerbators (clinical

characteristicsareshownintable1).Forbaselineanalysesofsputumsolublemediators(CXCL10/IP10,

MBL, SLPI and elafin proteins), samples from a total of 50 COPD patients (combined from the

communitycohortandfromsubjectsrecruitedtoexperimentalinfectionstudies)wereavailable,with

15patients(30%)classifiedasfrequentexacerbators(clinicalcharacteristicsarealsoshownintable

1).

Evaluationofbaselineexpressionofanti-viralmediators

Sincevirusesareamajorcauseofexacerbationsanddeficientanti-viralimmunityhasbeenobserved

in somebut not all studies of COPD 8-11,wehypothesised that frequent exacerbatorswouldhave

reducedbaselineexpressionofanti-viralimmunemediatorsandthusanimpairedpotentialtomount

protectiveresponsestovirusinfections.WeexaminedbaselinesputumcellmRNAexpressionoftype

IandIIIIFNsandISGsin36patientsfromthecommunity-basedCOPDcohortandfoundthatfrequent

exacerbatorshadsignificantlyreducedsputumIFNβandIFNλ2/3mRNAexpressionwithnodifference

inIFNλ1(Figure1a).Baselinesputumconcentrationsoftheinterferon-inducibleproteinCXCL10/IP-

10(Figure1b)werealsoreducedinfrequentexacerbatorsbutnodifferenceinmRNAexpressionof

otherISGs2’-5’OAS,viperinorMx1wasobserved(Figure1c).

SputuminterferonandISGexpressionduringviruspositiveexacerbation

Inthecommunity-basedcohort,18episodesofacuteexacerbationwithpositivevirusdetectionwere

observed(rhinovirusn=11,coronavirusn=4,RSVn=2,influenzan=1).Ofthese,n=7(38.9%)occurred

inpatientsclassifiedasfrequentexacerbators.Clinicalcharacteristicsoftheexacerbatingpatientsare

shownintable2.Onepatientwashospitalisedduringexacerbationwithallotherepisodestreatedin

the community. Having observed that frequent exacerbators have reduced capacity tomount an

innateanti-viralresponseatstablestate,wenextexaminedsputumcellmRNAexpressionofIFNsand

ISGs in the sub-group of patients who developed an exacerbation associated with positive virus

detection.FrequentexacerbatorshadsignificantlyreducedsputumcellexpressionofIFNβ,IFNλ2/3

andIFNλ1mRNAsatexacerbationpresentation(Figure2a).Sputum2’-5’OASmRNAexpressionwas

also significantly reducedatexacerbationpresentation in frequentversus infrequentexacerbators


https://doi.org/10.1101/632372

9

withnodifferencesobservedforviperinorMx1expression(Figure2b).Frequentexacerbatorsalso

had reduced sputum concentrations of the interferon-inducible cytokine CXCL10/IP-10 at

exacerbationonsetand2weekspost-onsetwithasimilarnon-significanttrend(P=0.25)observedat

exacerbationonsetforCCL5/RANTES(Figure2c).

Ex vivo bronchial epithelial anti-viral responses to RV infection are attenuated in frequent

exacerbators

Having observed reduced IFN- and ISG-expression in frequent exacerbators from in vivo sputum

samplestakenduringexacerbation,wenextproceededtodeterminewhetherepithelialcells from

patientswithCOPDwhoarefrequentexacerbatorshavedeficientinnateresponsestoRVinfection.

We utilised BECs collected bronchoscopically from sixteen patients with COPD (n=9 frequent

exacerbators,n=7infrequentexacerbators),differentiatedatanairliquidinterfaceandinfectedwith

RV-A1atMOI=0.1.Demographic/clinicalcharacteristicsoftheincludedpatientsareshowninTable

3.

Greater heterogeneity with trend for increased viral load was observed in BECs from frequent

exacerbators(SupplementaryFig1).SignificantinductionofIFNβandIFNλ1andIFNλ2/3mRNAsfrom

baselinewasobservedinbothfrequentandinfrequentexacerbatorsat72hourspost-infection(Figure

3a).RVinductionofIFNλ1mRNAwassignificantlylowerinfrequentversusinfrequentexacerbators

witha similar trend (P=0.29)observed for IFNβmRNAbutno significancedifferenceobserved for

IFNλ2/3(Fig3a).RVinductionoftheISGs2’-5’OASandViperinmRNAwasalsosignificantlylowerin

frequentversusexacerbatorswithnosignificantdifferenceobservedforPKR(Figure3b).

Significant inductionof IFN-β, IFN-λ1orIFN-λ2/3proteinsfrombaselinewasalsoobservedinboth

frequentandinfrequentexacerbatorsat72hourspost-infection(Figure3c).However,therewereno

significantdifferencesintheinductionofIFN-β,IFN-λ1orIFN-λ2/3proteinsbyRVbetweenfrequent

andinfrequentexacerbatorsat72hourspost-infection(Figure3c).

Collectively, these data supported our in vivo findings from exacerbating patients that anti-viral

responsesare reduced in frequentexacerbatorsandextended theseobservationsby identifyinga

dysregulatedinnateimmuneresponseinBECsdefinedbyimpairedexpressionofIFNandISGs


https://doi.org/10.1101/632372

10

Evaluationofsputumbacterialloadsatbaselineandduringvirus-associatedexacerbation

Virus-induced exacerbation can trigger subsequent bacterial infection in COPD12,13, a secondary

phenomenonthat isassociatedwith increasedexacerbationseverity12and inverselyrelatedtothe

interferonresponses21.Havingobservedthatanti-viralimmunityisreducedinfrequentexacerbators

bothinvivoandprimaryBECsfrompatientswithCOPDinvitro,wenextexaminedwhethersputum

bacterialloadsatstablestateandduringexacerbationdifferedaccordingtoexacerbationfrequency.

There were no differences in sputum 16S bacterial loads between frequent and infrequent

exacerbators atbaselineor exacerbationonset (Fig4a&b).However, frequentexacerbatorshada

significantincrease(~1log)inbacterialloadsat2weekspostexacerbationonset(Fig4c),suggesting

thatthisCOPDsubgroupmayhaveincreasedlikelihoodofsecondarybacterialinfectionduringvirus-

inducedexacerbation.

NodifferenceSLPIorelafinlevelsbetweenfrequentandinfrequentexacerbators

Wehavepreviously reported that rhinovirus-induced secondarybacterial infectionoccurs through

neutrophilelastase-mediatedcleavageandreductionoftheanti-microbialpeptides(AMPs)SLPIand

elafin,aprocess thatoccurs inCOPDbutnothealthysubjectsandmaybe furtheraccentuatedby

inhaled corticosteroid use21. Given our observation of increased bacterial loads in frequent

exacerbators,wenextexaminedwhetherthissub-groupofCOPDpatientshaveconcurrentlyreduced

SLPIandelafinlevels.TherewerenodifferencesinsputumSLPIorelafinlevelsbetweenfrequentand

infrequent exacerbators either at baseline (Supplementary Fig 2a-b) or during exacerbation

(SupplementaryFig1c-d).ThissuggestedthatdifferentialexpressionoftheseAMPsdoesnotunderlie

increasedsecondarybacterialinfectioninfrequentexacerbators.

Airwaymannose-bindinglectin2levelsarereducedinfrequentexacerbatorsatbaselineandduring

virus-associatedexacerbation

We next focused on other another AMP that could be important in driving secondary bacterial

infection inCOPD.Aprevious study reported thatmannose-binding lectin (MBL)-2polymorphisms

withassociatedMBL-2deficiencyinserumisassociatedwithrecurrenceofinfectiveexacerbationsin

COPD 25. Consistent with this report, we also found that MBL-2 concentrations in sputum were

significantlyreducedinfrequentversusinfrequentexacerbatorsatstablestate(Figure5a)andalsoat

2 weeks post exacerbation onset (Figure 5b), the same timepoint at which bacterial loads were

increased.


https://doi.org/10.1101/632372

11

Airway mannose-binding lectin 2 levels negatively correlates with bacterial loads during virus-

associatedexacerbation

HavingobservedthatfrequentexacerbatorshavereducedMBL-2levelsandincreasedbacterialloads

at 2weeks post exacerbation onset,we next examined the relationship between these variables.

TherewasasignificantnegativecorrelationbetweensputumconcentrationsofMBL-2andbacterial

loads(Figure5c)suggestingthatlowerMBL-2levelsmaycontributetoincreasedsecondarybacterial

infectioninfrequentexacerbators.

DISCUSSION

Theunderlyingmechanismsinvolvedinincreasedsusceptibilitytofrequentexacerbationsremainsa

crucialresearchquestioninCOPD.Weshowhere,forthefirsttime,thatpatientswithCOPDwhohave

ahistoryoffrequentexacerbationshavereducedairwayanti-microbialimmunitywhenassessedat

clinicalstabilityandduringsubsequentvirus-associatedexacerbationwithanassociatedincreasein

bacterial loads. We additionally confirm that COPD frequent exacerbators have reduced ex vivo

immune responses toRV infection inprimaryairwayepithelial cells, indicatingan innateanti-viral

deficiency. This is the first study to show that frequent exacerbators have impaired airway anti-

microbial responses and provides an importantmechanistic advance in our understanding of this

clinicallyimportantCOPDsub-group.Theseabnormalitiescouldpredisposesuchpatientstogreater

risk of acquisition of pathogenic viruses and bacteria and/or promote a greater likelihood of

developinganexacerbationfollowinginfection.

PreviousstudieshaveshownthatexvivotypeIIFNresponsestoRVinfectioninbronchoalveolarcells

andtoinfluenzainfectioninBECsareimpairedinCOPD8,9.PatientswithCOPDalsohaveincreased

virus loads and enhanced exacerbation severitywhen experimentally infectedwith rhinovirus 8,18.

However,defectsinanti-viralimmunityhavenotbeenshownuniversallywithotherstudiesreporting

theoppositeeffectofaugmentedexvivoanti-viralresponsesinCOPD10,11.Thediscrepancybetween

theseCOPDstudiessuggeststhatdefectiveimmunitymaynotbepresentinallpatientswithanumber

ofconfoundingcompetingfactorssuchasdiseaseseverityandmedicationslikelytohaveaninfluence.

Itisplausiblethatpatientswithfrequentexacerbationsmightrepresentonesub-groupwithimpaired

anti-viralimmunity.WereportherethatexpressionofbothtypeIandtypeIIIIFNs,IFNβandIFNλ2/3

mRNAsisreducedinfrequentexacerbatorsatstablestate,suggestingthatthesepatientsmighthave

areducedpotentialtogenerateprotectiveresponsestovirusinfection.Insupportofthishypothesis,

wealsoobservedthatanti-viralresponsesincludingIFNβ,IFNλ1andIFNλ2/3andtheISGs2’-5’OAS


https://doi.org/10.1101/632372

12

and CXCL10/IP-10 were suppressed during exacerbations in frequent exacerbators and that RV-

induction of IFNλ1, 2’-5’OAS and Viperin mRNAs in BECs ex vivo was also reduced in frequent

exacerbators.

Our study provides the first evidence that frequent exacerbators have innate anti-viral immune

dysfunction, suggesting that a reduction in interferon production might underlie an augmented

propensitytovirusinfectionsandthusincreasedexacerbationfrequencyobservedinthesepatients.

ThismechanismissupportedbyaclinicalstudywhichreportedthatCOPDpatientswhoarefrequent

exacerbatorsexperiencesignificantlymorecoryzalepisodesthan infrequentexacerbators26.Given

thatinhaledIFN-βtherapyhasbeenshowntoconferclinicalbenefitinasubgroupofsevereasthmatic

patientswhodevelopacold27,ourdataraisessuggestionthatsuchinnateimmune-boostingtherapies

couldbeeffectivewhenusedinatargetedmannerinpatientswithCOPDwithevidenceoffrequent

exacerbationsandalesseffectiveinnateimmuneresponse.

DespiteobservingimpairedexvivoRV-inductionofIFNandISGmRNAinfrequentexacerbatorBECs

at72hourspost-infection,wedidnotobservecorrespondingdifferencesinproteinproductionoftype

IandIIIIFNsbythesecellsatthesametimepoint.Ourstudieswerelimitedbysampleavailabilityonly

allowingevaluationatasingletimepoint.WehavepreviouslyreportedthatproteinproductionofIFN

persistsatleastupto96hoursinthiscelltype21anditisfeasiblethatevaluationatlatertimepoints

may be required to observe significant differences in translated IFN proteins. Despite a lack of

difference in absolute levels of IFN protein, the reduced ISG expression observed in frequent

exacerbatorssupportsalesseffectiveIFN-inducedresponseinthesesubjects

BacterialinfectionisalsoassociatedwithexacerbationinCOPDwithincreasedPCR-basedbacterial

detection at exacerbation versus stable state suggesting a causative role28,29. Additionally, virus-

inducedsecondarybacterialinfectionhasbeenreportedinbothexperimentalandnaturallyoccurring

exacerbations12,13.WehavepreviouslyreportedthatexperimentalRVchallengeinpatientswithCOPD

isassociatedwithincreasedfrequencyofsecondarybacterialinfectioncomparedtohealthysubjects12,30viaamechanismofneutrophilelastase-mediatedcleavageofanti-microbialpeptidesSLPIand

elafin12.Here,weextendthesefindingstorevealthatfrequentexacerbatorshavehigherbacterial

loadsat2weeksfollowingonsetofvirus-associatedexacerbation,suggestingthatthissubgroupof

COPDpatientsmightbeatgreatestriskofdevelopingsecondarybacterialinfectionfollowinginitial

virusinfection.AlthoughwefoundnodifferenceinlevelsofSLPIandelafinaccordingtoexacerbation

frequency, we did identify that expression of another AMP MBL-2 was reduced in frequent


https://doi.org/10.1101/632372

13

exacerbatorsatstablestateandduringviruspositiveexacerbation.ThepotentialimportanceofMBL-

2duringvirus-associatedexacerbationwasfurtherdemonstratedbyourfindingthatitsexpression

correlatednegativelywithbacterial loadsduring exacerbation.MBL is a key componentof innate

immunitythatactivatesthe lectincomplementpathwayand is involved intheprocessofbacterial

phagocytosis 31.MBLdeficiency is associatedwith susceptibility to respiratory tract infectionsand

pneumoniainhealthyindividuals32,33andtoincreasedriskofsepsisinanimalmodels34.MBLcanbind

to various pathogens of relevance to COPD exacerbations including both viruses and bacteria35.

GeneticMBL deficiency has been previously shown to be associatedwith both increased25,36 and

reduced37exacerbationfrequencyinCOPD.AlthoughserumMBLconcentrationshavebeenshownto

not differ between frequent and infrequent exacerbators36, a previous study reported reduced

bronchoalveolarlavageMBLconcentrationsinCOPDversushealthysubjects38.Ourdataextendsthese

observations by demonstrating that deficient airway MBL-2 at baseline and during subsequent

exacerbationmightpredisposefrequentexacerbatorsto increasedsecondarybacterial infection.A

previousstudyreportedthatthepresenceofrhinovirusisassociatedwithincreasedS.pneumoniae

colonization inchildrenwithMBLgenepolymorphisms, further suggestinga role forMBL invirus-

inducedsecondarybacterialinfection39.Giventhatnebulisedplasma-derivedMBLhasbeenshownto

restorephagocyticfunctioninsmoke-exposedmice40,ourdataraisespeculationthatadministration

ofMBLcouldadditionallybeconsideredasaneffectivepreventativeand/ortherapeuticstrategyfor

COPDexacerbations.Furtherstudiesareneededtoevaluatethis.

Itisimportanttonotethatcausationcannotbeinferredfromtheresultsofourstudy.Althoughwe

found reduced anti-microbial responses in patients with a history of frequent exacerbations, we

cannotreliableconcludethattheseabnormalitiesdirectlyleadtoinherentincreasedexacerbationrisk

perse.Therearelikelytobeanumberofadditionalfactorsthatmaybecontributingtotheobserved

differences and, notably, in stable state analyses, frequent exacerbators showed trends towards

greaterICSuseandlesscurrentsmoking.Theseconfounderscouldcontributedirectlyorindirectlyto

the differences observed, as previously reported21,41,42. We therefore consider our study to be

hypothesisgeneratingandemphasisetheimportanceoffuturelargerinvivostudiestomorecarefully

dissecttheinfluenceofvariousfactorsanddefinitelyanswerthequestionofwhetherdeficientanti-

microbial immunityunderliespropensitytoexacerbationfrequencyinCOPD.Previousstudieshave

also indicated that exacerbation risk inCOPD is dynamic andmay change fromyear to year4,43. A

longer-term studywith repeated airway samplingwould be need to determinewhether baseline

antimicrobialresponsesaresimilarlydynamicinnature.


https://doi.org/10.1101/632372

14

Inconclusion,weshowthatpatientswithCOPDandahistoryoffrequentexacerbationshavereduced

anti-viralandanti-bacterialimmunityassociatedwithincreasedsecondarybacterialinfection.These

immunedefectsmayunderlietheincreasedpropensitytoexacerbationsobservedinthissub-group

andprovidesevidencetosupporttherapeuticapproachesthatboostinnateimmunityinCOPD


https://doi.org/10.1101/632372

15

TABLESPatientsincludedinbaselinemRNAanalyses(n=36) Frequent

exacerbators(n=13)

Infrequentexacerbators(n=23)

Pvalue

Malesex 10(66.7%) 27(77.1%) 0.49Age 68.5(58.3-75.3) 67(59.0-71.5) 0.69GOLDstageI/II/III/IV 0/9/3/3 5/28/2/0

Longtermoxygenuse 1(6.7%) 0(0%) 0.30Cardiacdisease 4(26.7%) 2(5.7%) 0.058Cerebrovasculardisease 3(20%) 1(2.9%) 0.075Diabetes 2(13.3%) 2(5.7%) 0.57Pulmonaryhypertension

0(0%) 1(2.9%) 1.0

Inhaled corticosteroiduse

9(56.3%) 11(31.4%) 0.11

Betaagonistuse 14(93.3%) 18(51.4%) 0.0046

Anti-muscarinic inhaleruse

12(80.0%) 12(34.3%) 0.0049

Currentsmoker 6(40.0%) 7(20.0%) 0.17Patientsincludedinbaselinesputumsolublemediatoranalyses(n=50) Frequent

exacerbators(n=15)

Infrequentexacerbators(n=35)

Pvalue

Malesex 10(66.7%) 27(77.1%) 0.49Age 68.5(58.3-75.3) 67(59.0-71.5) 0.69GOLDstageI/II/III/IV 0/9/3/3 5/28/2/0 Longtermoxygenuse 1(6.7%) 0(0%) 0.30Cardiacdisease 4(26.7%) 2(5.7%) 0.058Cerebrovasculardisease 3(20%) 1(2.9%) 0.075Diabetes 2(13.3%) 2(5.7%) 0.57Pulmonaryhypertension

0(0%) 1(2.9%) 1.0

Inhaled corticosteroiduse

9(56.3%) 11(31.4%) 0.11

Betaagonistuse 14(93.3%) 18(51.4%) 0.0046Anti-muscarinic inhaleruse

12(80.0%) 12(34.3%) 0.0049

Currentsmoker 6(40.0%) 7(20.0%) 0.17

Table1:DemographicandclinicalcharacteristicsoffrequentandinfrequentexacerbatorsincludedinbaselinemRNAanalysesandsputumsolublemediatoranalyses.GOLD=GlobalInitiativeforobstructivelungdisease


https://doi.org/10.1101/632372

16

Frequentexacerbators

(n=7)Infrequentexacerbations(n=11)

Pvalue

Age(median(IQR))

69(64-74) 71(52-76) 0.84

Malesex 3(42.9%) 9(81.8%) 0.14GOLDStage(median(IQR))

2(2-3) 2(1-2) 0.13

Longtermoxygen 0(0%) 0(0%) 1.0Cardiacdisease 0(0%) 1(9.1%) 1.0Cerebrovasculardisease

1(14.3%) 0(0%) 0.39

Diabetes 1(14.3%) 1(9.1%) 1.0Pulmonaryhypertension

0(0%) 0(0%) 1.0

Inhaledcorticosteroiduse

4(57.1%) 3(27.3%) 0.33

Betaagonistuse 6(85.7%) 7(63.6%) 0.60Anti-muscarinicinhaleruse

4(57.1%) 6(54.5%) 1.0

Currentsmoker 4(57.1%) 2(18.2%) 0.14TreatmentsduringexacerbationAntibioticsOralcorticosteroids

5(71.1%)3(42.9%)

3(27.3%)0(0%)

0.150.043

Table2:Characteristicsofpatientswhodevelopedvirusassociatedexacerbationstratifiedaccordingtoexacerbationfrequency.


https://doi.org/10.1101/632372

17

Frequentexacerbators(n=9)

Infrequentexacerbations(n=7)

Pvalue

Age

77(72.5-82) 67(63.5–71) 0.29

GOLDstage23

1(11.1%)8(88.9%)

2(28.6%)6(85.7%)

0.551.0

Malesex 6(66.7%) 3(42.9%) 0.61Inhaledcorticosteroiduse

7(77.8%) 3(42.9% 0.30

LongactingBetaagonistuse

7(77.8%) 3(42.9%) 0.30

Anti-muscarinicinhaleruse

7(77.8%) 3(42.9%) 0.30

Currentsmoker 1(11.1%) 3(42.9%) 0.26Table3:BaselinecharacteristicsofpatientswithCOPDundergoingbronchoscopicsamplingforairwayepithelialcellexperimentsshowninfigure5.


https://doi.org/10.1101/632372

18

FIGURESLEGENDS

Figure1:COPDpatientswithfrequentexacerbationshavereducedanti-viral immunityatclinicalstability.AcohortofpatientswithCOPDweremonitoredprospectivelyandsputumsamplesweretakenwhenclinicallystableforatleast6weeks.Patientswerestratifiedaccordingtoexacerbationfrequencyintheprecedingyearwithpatientswhoexperienced>2exacerbationepisodesclassifiedas ‘frequent’. Sputum cell mRNA expression of (a) IFNβ, IFNλ2/3 and IFNλ1 was measured byquantitativePCR.(b)SputumsupernatantproteinconcentrationsofCXCL10/IP-10weremeasuredbyELISA.SputumcellmRNAexpressionof interferon-stimulatedgenes(ISGs)(c)2’-5’OAS,viperinandMx1wasmeasuredbyquantitativePCR.Boxandwhiskerplotsshowingmedian(linewithinbox),IQR(box)andminimumtomaximum(whiskers).StatisticalcomparisonsweremadeusingMannWhitneyUtest.*p<0.05.ns=non-significant.


https://doi.org/10.1101/632372

19

Figure2:COPDpatientswithfrequentexacerbationshavereducedanti-viralimmunityduringvirus-associatednaturallyoccurringexacerbations.AcohortofpatientswithCOPDweremonitoredprospectively.Patientswerestratifiedaccordingtoexacerbationfrequencyintheprecedingyearwithpatientswhoexperienced>2exacerbationepisodesclassifiedas‘frequent’.Sputumsamplesweretakenatpresentationwithexacerbationassociatedwithpositivevirusdetectionand2weeksduringexacerbation.SputummRNAexpressionof(a)IFNβ,IFNλ2/3andIFNλ1(b)2’-5’OAS,viperinandMx1wasmeasuredbyquantitativePCR.Sputumproteinconcentrationsof(c)CXCL10/IP-10andCCL5/RANTESweremeasuredbyELISA.Datadisplayedasboxandwhiskerplotsshowingmedian(linewithinbox),IQR(box)andminimumtomaximum(whiskers).StatisticalcomparisonsweremadeusingMannWhitneyUtest.*p<0.05.ns=non-significant.


https://doi.org/10.1101/632372

20

Figure3:COPDpatientswithfrequentexacerbationshavedeficientexvivoinnateanti-viralimmuneresponsestorhinovirusinfectionindifferentiatedairwayepithelialcells.Primarybronchialepithelialcells (BECs) from 16 patients with GOLD stage II or III COPDwere differentiated at the air-liquidinterfaceandinfectedexvivowithrhinovirus(RV)-A1ormediumcontrol(baseline).Celllysatesandsupernatantswereharvestedpost-infection.(a)IFNβ,IFNλ1andIFNλ2/3(b)2’-5’OAS,PKRandviperinmRNAexpressionincelllysatesat72hwasmeasuredbyquantitativePCR.(c)IFN-β,IFN-λ1andIFNλ-2/3proteinsweremeasuredat72hincellsupernatantsbyELISA.DatarepresentsindividualpatientsandanalysedbyWilcoxonranksumtest(baselineversusRV)orMannWhitneyUtest(RVfrequentexacerbatorsversusRVinfrequentexacerbator).*p<.05;**p<0.01.ns=non-significant.


https://doi.org/10.1101/632372

21

Figure4:COPDpatientswith frequentexacerbationshave increasedbacterial loadsduringvirusassociated naturally occurring exacerbations. A cohort of patients with COPD were monitoredprospectively.Patientswerestratifiedaccordingtoexacerbationfrequencyintheprecedingyearwithpatientswhoexperienced>2exacerbationepisodesclassifiedas‘frequent’.Sputumsamplesweretakenatpresentationwithexacerbationassociatedwithpositivevirusdetectionand2weeksduringexacerbation. Sputum bacterial loads weremeasured by 16S quantitative PCR at (a) baseline (b)exacerbationand(c)2weeks.Datashownasmedian(IQR)andanalysedbyMannWhitneyUtest.*p<0.05.ns=non-significant.


https://doi.org/10.1101/632372

22

Figure5:COPDpatientswithfrequentexacerbationshavereducedairwaymannose-bindinglectin2 levels which correlate negatively with bacterial loads during virus associated exacerbations.Sputumproteinconcentrationsofmannosebindinglectinat(a)stablestate(b)duringvirusassociatedexacerbation.(c)Correlationofsputumbacterialloadswithsputumproteinlevelsofmannose-bindinglectin(MBL)-2.In(a)&(b)datadisplayedasboxandwhiskerplotsshowingmedian(linewithinbox),IQR(box)andminimumtomaximum(whisker)andstatisticalcomparisonsweremadeusingMannWhitneyUtest.In(c),correlationanalysisusedwasnonparametric(Spearman'scorrelation).*p<0.05.ns=non-significant.


https://doi.org/10.1101/632372

23

References1. SeemungalTA,DonaldsonGC,PaulEA,BestallJC,JeffriesDJ,WedzichaJA.Effectof

exacerbationonqualityoflifeinpatientswithchronicobstructivepulmonarydisease.

Americanjournalofrespiratoryandcriticalcaremedicine.1998;157(5Pt1):1418-1422.

2. DonaldsonGC,SeemungalTA,BhowmikA,WedzichaJA.Relationshipbetween

exacerbationfrequencyandlungfunctiondeclineinchronicobstructivepulmonarydisease.

Thorax.2002;57(10):847-852.

3. SinganayagamA,SchembriS,ChalmersJD.Predictorsofmortalityinhospitalized

adultswithacuteexacerbationofchronicobstructivepulmonarydisease.Annalsofthe

AmericanThoracicSociety.2013;10(2):81-89.

4. HurstJR,VestboJ,AnzuetoA,etal.Susceptibilitytoexacerbationinchronic

obstructivepulmonarydisease.TheNewEnglandjournalofmedicine.2010;363(12):1128-

1138.

5. Soler-CatalunaJJ,Martinez-GarciaMA,RomanSanchezP,SalcedoE,NavarroM,

OchandoR.Severeacuteexacerbationsandmortalityinpatientswithchronicobstructive

pulmonarydisease.Thorax.2005;60(11):925-931.

6. SeemungalT,Harper-OwenR,BhowmikA,etal.Respiratoryviruses,symptoms,and

inflammatorymarkersinacuteexacerbationsandstablechronicobstructivepulmonary

disease.Americanjournalofrespiratoryandcriticalcaremedicine.2001;164(9):1618-1623.

7. RohdeG,WiethegeA,BorgI,etal.Respiratoryvirusesinexacerbationsofchronic

obstructivepulmonarydiseaserequiringhospitalisation:acase-controlstudy.Thorax.

2003;58(1):37-42.

8. MalliaP,MessageSD,GielenV,etal.Experimentalrhinovirusinfectionasahuman

modelofchronicobstructivepulmonarydiseaseexacerbation.AmJRespirCritCareMed.

2011;183(6):734-742.

9. HsuAC,ParsonsK,MoheimaniF,etal.ImpairedAntiviralStressGranuleandIFN-

betaEnhanceosomeFormationEnhancesSusceptibilitytoInfluenzaInfectioninChronic


https://doi.org/10.1101/632372

24

ObstructivePulmonaryDiseaseEpithelium.Americanjournalofrespiratorycelland

molecularbiology.2016;55(1):117-127.

10. BainesKJ,HsuAC,ToozeM,GunawardhanaLP,GibsonPG,WarkPA.Novelimmune

genesassociatedwithexcessiveinflammatoryandantiviralresponsestorhinovirusinCOPD.

Respiratoryresearch.2013;14:15.

11. KanOK,RamirezR,MacDonaldMI,etal.HumanMetapneumovirusInfectionin

ChronicObstructivePulmonaryDisease:ImpactofGlucocorticosteroidsandInterferon.The

Journalofinfectiousdiseases.2017;215(10):1536-1545.

12. MalliaP,FootittJ,SoteroR,etal.Rhinovirusinfectioninducesdegradationof

antimicrobialpeptidesandsecondarybacterialinfectioninchronicobstructivepulmonary

disease.AmJRespirCritCareMed.2012;186(11):1117-1124.

13. GeorgeSN,GarchaDS,MackayAJ,etal.Humanrhinovirusinfectionduringnaturally

occurringCOPDexacerbations.TheEuropeanrespiratoryjournal:officialjournalofthe

EuropeanSocietyforClinicalRespiratoryPhysiology.2014.

14. PatelIS,SeemungalTA,WilksM,Lloyd-OwenSJ,DonaldsonGC,WedzichaJA.

Relationshipbetweenbacterialcolonisationandthefrequency,character,andseverityof

COPDexacerbations.Thorax.2002;57(9):759-764.

15. PizzichiniE,PizzichiniMM,LeighR,DjukanovicR,SterkPJ.Safetyofsputum

induction.TheEuropeanrespiratoryjournal.Supplement.2002;37:9s-18s.

16. VogelmeierCF,CrinerGJ,MartinezFJ,etal.GlobalStrategyfortheDiagnosis,

Management,andPreventionofChronicObstructiveLungDisease2017Report.GOLD

ExecutiveSummary.Americanjournalofrespiratoryandcriticalcaremedicine.

2017;195(5):557-582.

17. MalliaP,WebberJ,GillSK,etal.Roleofairwayglucoseinbacterialinfectionsin

patientswithchronicobstructivepulmonarydisease.TheJournalofallergyandclinical

immunology.2017.


https://doi.org/10.1101/632372

25

18. FootittJ,MalliaP,DurhamAL,etal.OxidativeandNitrosativeStressandHistone

Deacetylase-2ActivityinExacerbationsofCOPD.Chest.2016;149(1):62-73.

19. HackettTL,SingheraGK,ShaheenF,etal.Intrinsicphenotypicdifferencesof

asthmaticepitheliumanditsinflammatoryresponsestorespiratorysyncytialvirusandair

pollution.AmJRespirCellMolBiol.2011;45(5):1090-1100.

20. HackettTL,WarnerSM,StefanowiczD,etal.Inductionofepithelial-mesenchymal

transitioninprimaryairwayepithelialcellsfrompatientswithasthmabytransforming

growthfactor-beta1.Americanjournalofrespiratoryandcriticalcaremedicine.

2009;180(2):122-133.

21. SinganayagamA,GlanvilleN,GirkinJL,etal.Corticosteroidsuppressionofantiviral

immunityincreasesbacterialloadsandmucusproductioninCOPDexacerbations.Nat

Commun.2018;9(1):2229.

22. BartlettNW,WaltonRP,EdwardsMR,etal.Mousemodelsofrhinovirus-induced

diseaseandexacerbationofallergicairwayinflammation.NatMed.2008;14(2):199-204.

23. SlaterL,BartlettNW,HaasJJ,etal.Co-ordinatedroleofTLR3,RIG-IandMDA5inthe

innateresponsetorhinovirusinbronchialepithelium.PLoSpathogens.

2010;6(11):e1001178.

24. CoxMJ,TurekEM,HennessyC,etal.Longitudinalassessmentofsputummicrobiome

bysequencingofthe16SrRNAgeneinnon-cysticfibrosisbronchiectasispatients.PloSone.

2017;12(2):e0170622.

25. LinCL,SiuLK,LinJC,etal.Mannose-bindinglectingenepolymorphismcontributesto

recurrenceofinfectiveexacerbationinpatientswithCOPD.Chest.2011;139(1):43-51.

26. HurstJR,DonaldsonGC,WilkinsonTM,PereraWR,WedzichaJA.Epidemiological

relationshipsbetweenthecommoncoldandexacerbationfrequencyinCOPD.EurRespirJ.

2005;26(5):846-852.


https://doi.org/10.1101/632372

26

27. DjukanovicR,HarrisonT,JohnstonSL,etal.TheeffectofinhaledIFN-betaon

worseningofasthmasymptomscausedbyviralinfections.Arandomizedtrial.AmJRespir

CritCareMed.2014;190(2):145-154.

28. SethiS,EvansN,GrantBJ,MurphyTF.Newstrainsofbacteriaandexacerbationsof

chronicobstructivepulmonarydisease.TheNewEnglandjournalofmedicine.

2002;347(7):465-471.

29. GarchaDS,ThurstonSJ,PatelAR,etal.Changesinprevalenceandloadofairway

bacteriausingquantitativePCRinstableandexacerbatedCOPD.Thorax.2012;67(12):1075-

1080.

30. MolyneauxPL,MalliaP,CoxMJ,etal.Outgrowthofthebacterialairwaymicrobiome

afterrhinovirusexacerbationofchronicobstructivepulmonarydisease.Americanjournalof

respiratoryandcriticalcaremedicine.2013;188(10):1224-1231.

31. FidlerKJ,HilliardTN,BushA,etal.Mannose-bindinglectinispresentintheinfected

airway:apossiblepulmonarydefencemechanism.Thorax.2009;64(2):150-155.

32. RantalaA,LajunenT,JuvonenR,etal.Mannose-bindinglectinconcentrations,MBL2

polymorphisms,andsusceptibilitytorespiratorytractinfectionsinyoungmen.TheJournal

ofinfectiousdiseases.2008;198(8):1247-1253.

33. EndemanH,HerpersBL,deJongBA,etal.Mannose-bindinglectingenotypesin

susceptibilitytocommunity-acquiredpneumonia.Chest.2008;134(6):1135-1140.

34. ShiL,TakahashiK,DundeeJ,etal.Mannose-bindinglectin-deficientmiceare

susceptibletoinfectionwithStaphylococcusaureus.JExpMed.2004;199(10):1379-1390.

35. NethO,JackDL,DoddsAW,HolzelH,KleinNJ,TurnerMW.Mannose-bindinglectin

bindstoarangeofclinicallyrelevantmicroorganismsandpromotescomplement

deposition.Infectionandimmunity.2000;68(2):688-693.

36. MandalJ,MallaB,SteffensenR,etal.Mannose-bindinglectinproteinandits

associationtoclinicaloutcomesinCOPD:alongitudinalstudy.Respiratoryresearch.

2015;16:150.


https://doi.org/10.1101/632372

27

37. DickerAJ,CrichtonML,CassidyAJ,etal.Geneticmannosebindinglectindeficiencyis

associatedwithairwaymicrobiotadiversityandreducedexacerbationfrequencyinCOPD.

Thorax.2017.

38. HodgeS,HodgeG,JersmannH,etal.Azithromycinimprovesmacrophagephagocytic

functionandexpressionofmannosereceptorinchronicobstructivepulmonarydisease.

Americanjournalofrespiratoryandcriticalcaremedicine.2008;178(2):139-148.

39. KarppinenS,VuononvirtaJ,HeQ,WarisM,PeltolaV.EffectsofRhinovirusInfection

onNasopharyngealBacterialColonizationinInfantsWithWildorVariantTypesofMannose-

BindingLectinandToll-LikeReceptors3and4.JPediatricInfectDisSoc.2013;2(3):240-247.

40. HodgeS,MatthewsG,DeanMM,etal.Therapeuticroleformannose-bindinglectin

incigarettesmoke-inducedlunginflammation?Evidencefromamurinemodel.American

journalofrespiratorycellandmolecularbiology.2010;42(2):235-242.

41. ThomasBJ,PorrittRA,HertzogPJ,BardinPG,TateMD.Glucocorticosteroidsenhance

replicationofrespiratoryviruses:effectofadjuvantinterferon.Scientificreports.

2014;4:7176.

42. EddlestonJ,LeeRU,DoernerAM,HerschbachJ,ZurawBL.Cigarettesmoke

decreasesinnateresponsesofepithelialcellstorhinovirusinfection.Americanjournalof

respiratorycellandmolecularbiology.2011;44(1):118-126.

43. HanMK,QuibreraPM,CarrettaEE,etal.Frequencyofexacerbationsinpatientswith

chronicobstructivepulmonarydisease:ananalysisoftheSPIROMICScohort.TheLancet.

Respiratorymedicine.2017;5(8):619-626.


https://doi.org/10.1101/632372

28

Acknowledgements:Funding:WellcomeTrustClinicalResearchTrainingFellowship(grantnumberWT096382AIA)ImperialCollegeHealthcareTrustBiomedicalResearchCentregrant(P33132);AcademyofMedicalSciencesand Wellcome Trust Starter Grant; Medical Research Council Program Grant G0600879; BritishMedical Association H.C. Roscoe Fellowships; BLF/Severin Wunderman Family Foundation LungResearchProgramGrantP00/2; Imperial CollegeandNIHRBRC funding scheme; theNIHRClinicalLecturerfundingscheme

Authorcontributions:

ConceptionandDesign:AS,SLL,JF,SLJ,NWB,PM

Experimentalwork:AS,SLL,MC,MT,LJF,EB,JG,PV,PSP,KSN,AR,JF,NWB,PM

Writingofmanuscript:Allauthors

Guarantor:AS


https://doi.org/10.1101/632372

Documents

Anti-microbial immunity is impaired in COPD patients with ... · an upper respiratory tract infection or an increase in any of the symptoms of dyspnoea, cough, sputum volume or purulence