differential expression of soluble receptor for …...2019/08/05 · 21 s. m. cruickshank, av hill...

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Differentialexpressionofsolublereceptorforadvancedglycationend-products(sRAGE)in1

micesusceptibleorresistanttochroniccolitis2

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Michael Bramhall1,2, Kevin Rich2, Ajanta Chakraborty2, Larisa Logunova2, Namshik Han3,4,4

JamesWilson5,CatherineBooth5,JohnMclaughlin3,6,AndyBrass3,SheenaM.Cruickshank25

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AuthorAffiliations:7

1DepartmentofBiochemistryandMolecularBiology,SchoolofBiomedicalSciences,Monash8

University,Clayton,Victoria3800,Australia9

2Lydia Becker Institute of Immunology and Inflammation, Manchester Academic Health10

Science Centre, School of Biological Sciences, Faculty of Biology, Medicine, and11

Health,UniversityofManchester,ManchesterM139PT,UK12

3ManchesterAcademicHealthScienceCentre,SchoolofMedicalSciences,TheUniversityof13

Manchester,ManchesterM139PT,UK14

4TheGurdonInstitute,UniversityofCambridge,CambridgeCB21QN,UK15

5EpistemLtd.,ManchesterM139XX,UK16

6SalfordRoyalNHSFoundationTrust,SalfordM68HDUK17

Runningtitle:Markersofprotectiveresponsesincolitis18

19

CorrespondingAuthor20

S.M.Cruickshank,AVHillBuilding,UniversityofManchester,ManchesterM139PT,UK21

[email protected], Phone+44(0)1612751578 22

not certified by peer review) is the author/funder. All rights reserved. No reuse allowed without permission. The copyright holder for this preprint (which wasthis version posted August 5, 2019. ; https://doi.org/10.1101/719310doi: bioRxiv preprint

https://doi.org/10.1101/719310

Abstract23

Aims: Identifying the factors that contribute to chronicity in inflamed colitic tissue is not24

trivial.However,inmousemodelsofcolitis,wecaninvestigateatpreclinicaltimepoints.We25

sought tovalidatemurineTrichurismuris infectionasamodel for identificationof factors26

thatpromotedevelopmentofchroniccolitis.27

Methods:WecomparedpreclinicalchangesinmicewitharesolvingimmuneresponsetoT.28

muris (resistant) versus mice that fail to expel the worms and develop chronic colitis29

(susceptible).Findingswerethenvalidatedinhealthycontrolsandpatientswithsuspected30

orconfirmedIBD.31

Results:TheReceptorforAdvancedGlycationEndProducts(Rage)washighlydysregulated32

between resistant and susceptible mice prior to the onset of any pathological signs.33

IncreasedsolubleRAGE(sRAGE) intheserumandfaecesofresistantmicecorrelatedwith34

reducedcolitisscores.Mousemodelfindingswerevalidatedinapreliminaryclinicalstudy:35

faecalsRAGEwasdifferentiallyexpressedinpatientswithactiveIBDcomparedwithIBDin36

remission,patientswithIBDexcludedorhealthycontrols.37

Conclusion: Pre-clinical changes in mouse models can identify early pathways in the38

developmentofchronicinflammationthathumanstudiescannot.Weidentifiedthedecoy39

receptor sRAGE as a potentialmechanism for protection against chronic inflammation in40

colitisinmiceandhumans.WeproposethattheRAGEpathwayisclinicallyrelevantinthe41

onset of chronic colitis, and that further study of sRAGE in IBD may provide a novel42

diagnosticandtherapeutictarget.43

44

Keywords:sRAGE,colitis,mouse45

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Introduction47

Inflammatory bowel diseases (IBD) are a group of intestinal immune disorders, including48

Crohn’sdisease(CD)andulcerativecolitis(UC),thatcausechronicinflammationinthegut49

[1]. The cause of IBD is currently not known, but dysregulation of intestinal immunity,50

microbial dysbiosis, genetics and environmental factors contribute to disease onset.51

Unpredictablecyclesofremissionandrelapserequirecarefulmonitoringandthelong-term52

damage from inflammationoftenwarrantspotent immunomodulatory therapyor surgical53

intervention[2].54

Itisimpossibletoreliablypredictonset,relapseorremissionofIBD[3]andcurrently,only55

animalmodelsprovideameansofstudyingtheperturbationsinthegutthatprecedecolitis.56

Infecting susceptible mouse strains with the enteric nematode parasite Trichuris muris57

closelyparallelshumanCrohn’sdiseaseinboththepathologicalandtranscriptionalchanges58

induced[4]andhasbeenestablishedasamodelforthestudyoftheinitiationof immune59

responses in the colon [5]. T. muris resistant BALB/c and C57BL6 mice mount an early60

immune response against thewormswithin 24 hours of infection,with large numbers of61

dendritic cells (DCs)migrating to the lamina propria,whereasAKRmice or C57BL/6mice62

withalowdoseinfectionmountadelayedimmuneresponse,resultinginchronicintestinal63

inflammationandafailuretoexpeltheworms[5,6].Bothsusceptibleandresistantstrains64

showmildsignsofinflammationwithin24hours.However,inflammationinresistantmiceis65

controlled and ultimately resolves, whereas susceptible strains go on to develop clinical66

colitisinthesubsequentweekspostinfection.67

Exploiting these early differences in the host immune response to T. muris infection68

experimentallymayprovideinformationonthefactorsthatpromotetheonsetofchronic,69

ratherthanresolving,inflammationinthegut[7].Earlyfactorsareimpossibletodistinguish70


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from the inflammatorymilieupresent in IBDpatients at thepointofdiagnosis as chronic71

inflammation is already well established. Identification and validation of early changes72

duringchroniccolitisonsetinmicecouldprovideausefulpipelinefordevelopingdiagnostic73

anddisease-managementbiomarkersortherapeutictargetsinhumancolitis.74

In this study, we carried out a T. muris infection study, investigating preclinical75

transcriptionalchanges24hourspostinfection(PI).Weidentifiedthereceptorforadvanced76

glycation end-products (Rage) as highly upregulated in mice susceptible to T. muris77

infection.WefurtherinvestigatedthepresenceofRAGEandrelatedligandsincoliticmice78

andcarriedoutatranslationalvalidationstudy investigatingthepresenceofsolubleRAGE79

(sRAGE)inthefaecesofIBDpatientsandhealthycontrols.80

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Materialsandmethods82

Mice83

All animal procedures used in this project were carried out in accordance with the UK84

Animals(ScientificProcedures)Act,1986.ForT.muris infectionexperiments,6-8weekold85

male BALB/c and AKR mice were used (Harlan UK, Bicester, UK). Mice were housed in86

individuallyventilatedcageswithnestingmaterialandweremaintainedunderconstant12h87

light–darkcycleat21-23°Cwithfreeaccesstowaterandstandardchow(BeekayRatand88

Mouse Diet, Bantin & Kingham, Hull, UK). Euthanasia was carried out by schedule 189

procedureofCO2asphyxiationfollowedbycervicaldislocationorexsanguination.3-6mice90

wereusedperstrain,pertimepointstudied.91

Parasitesandinfection92

Professor Kathryn Else, The University of Manchester, kindly provided eggs of T. muris93

Edinburgh (E) isolate for use in all infection studies. Egg infectivity and maintenance of94

parasite stocks were carried as described byWakelin, 1967 [8]. Experimental mice were95

infectedwith200embryonatedeggsin200μlofultra-puredistilledwaterviaoralgavage.96

Wormburdenwas assessed at day 21 PI. Caecum and proximal colonwere harvested at97

autopsytodetermineparasiteclearanceofeachmouseat theendofeachexperimentas98

describedbyElseetal.,1990[9].99

Humansamples100

Prior to thecommencementof theclinical study,NHSethicsapprovalwasobtained from101

BerkshireBResearchEthicsCommittee(RECreferencenumber:14/SC/1413;IRASreference102

number:157778) inordertoscreenclinical IBDsamplesof faecesandserum.Participants103

were recruited from the Salford Royal NHS Foundation Trust. Normal healthy volunteers104

wererecruited inaccordancewith theUniversityethicscommitteeandtheHumanTissue105


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Act2004.Faecalsamplesweretakenfromhealthycontrols(n=10)withnopriorhistoryof106

IBDorgutproblems,orpatients (n=31)withsuspected IBDorclinicallyconfirmed IBD.All107

patientsamplesweretakenviaoutpatientclinics,returnedbypatientsaspartofstandard108

clinical practice to be assessed for Faecal Calprotectin (FCP). Colonoscopy/biopsy were109

undertakeninthosewithelevatedFCP.Ofthepatients,6patientshadIBDexcluded,mainly110

leadingtoaclinicaldiagnosisofirritablebowelsyndrome(IBS),19knownIBDpatientswere111

inremissionattheoftimetesting(10ulcerativecolitisand9Crohn’sdisease)and6patients112

hadactiveIBD(n=5CD,n=1UC)atthetimeoftesting.113

Statisticsandanalysis114

Where statistics arequoted, experimental groupswere comparedusing linear regression,115

Mann-WhitneyUtestortwo-wayanalysisofvariance(ANOVA)testfollowedbySidak’spost116

hoc multiple comparisons test, where appropriate. P values <0.05 were considered117

significant.Dataarepresentedasmean±SEMunlessotherwisestated.Statisticalanalyses118

were carried out using GraphPad Prism 7 (GraphPad Software, La Jolla, California, USA;119

www.graphpad.com). 120


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Results121

EarlyimmuneresponseinformsresistancetoT.muris-inducedcolitis122

Following challengewithTrichurismuris, asexpectedBALB/cmiceexpelledmostorallof123

thewormsby21daysPI,whereasAKRmicewereunabletoexpelallwormsandremained124

infectedwith a significantly higherwormburden (P=0.016,Mann-WhitneyU test) (Figure125

1A).Colitisscoringrevealedincreasedhistologicalchangesassociatedwithinflammationin126

both AKR and BALB/c mice after infection (Figure 1B). These changes included influx of127

immunecells,presenceofimmunecellsinthesubmucosa,crypthyperplasiaandgobletcell128

loss.Inagreementwithpreviousdata,thecolitisscoresinBALB/cmicepeakedat21daysPI129

andhadbegun to return tonormalby31daysPI.Asexpected, colitis scores inAKRmice130

roseafterinfectionandpeakedat31daysPIwherethecolitisscorewassignificantlygreater131

than that of the BALB/c mice (P=0.046, ANOVA; Figure 1B). Representative images of132

haematoxylinandeosinstainedproximalcolonsectionsinnaïvemiceandat31daysPIare133

shown in Figure1C. Collectively, these results reproducepreviouslypublished research in134

the AKR/BALB/c infection model [4], where BALB/c mice initiate an acute, resolving135

inflammationafterT.murischallengeandAKRmice showdelayed immune response that136

resultsinachronicinflammatoryphenotypeduetoafailuretoexpelworms.137

Weinvestigatedimmunecellrecruitmenttothecoloniclaminapropriabyflowcytometryat138

1,7,14,21and31daysPI.BALB/cmicehadanacuteresolvingresponsewhereasAKRmice139

developedachronicinflammatoryresponse.At24hoursPIBALB/cmicerespondedrapidly140

toT.muris challenge,withanearly increase in theproportionsofDCs,macrophagesand141

neutrophils(P<0.05,ANOVA)incoloniclaminapropriatissuesandmesentericlymphnodes142

compared to naïve mice (Figure 1D and 1E, Supplementary data). BALB/c and AKR had143

differentresponsestoinfection,withBALB/ctendingtohavegreaterrecruitmentofinnate144


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immune cells at D1 compared with susceptible AKR mice. This trend for a greater early145

magnitudeofresponseintheBALB/ccomparedwithAKRwasalsoseenatD7,D14andD21146

post-infectionwiththegreatestdifferencesbetweentheimmuneresponseofBALB/cmice147

to AKR at D14 PI (Figure 1B, Supplementary data). However, by D31, proportions of148

macrophages(P<0.001),inflammatorymonocytes(P<0.01)andneutrophils(P<0.001),were149

all significantlygreater inAKRmice,whereas theproportionsof thesecell types returned150

neartobaselinelevelsinBALB/cmice(Figure1D-G).Theincreaseinimmunecellsobserved151

inAKRmiceatD31correspondedwiththepeakcolitisscore,andlikewisethereductionin152

immunecellsinBALB/cmiceatD31wasparalleledwithareductionincolitisscore(Figure153

1B). Collectively thiswork indicated an altered dynamic of immune response in resistant154

versus susceptible mice, therefore we explored the early transcriptome in order to155

understandchangespresentbetweencolitis-susceptibleandcolitis-resistantmice.156

TranscriptionalchangesinducedbyT.murisinfectionidentifiedat24hourspostinfection157

Transcriptionalchangesintheproximalcolon(theprincipalsiteofT.muris infection)were158

investigatedat24hourspost-infectionviamicroarray,prior to theestablishmentofovert159

signsofinflammation.Geneswiththelargestdifferentialexpressionat24hourspostwere160

calculated(Figure2A).Ofthe77probesetsthatweresignificantlyupregulatedinAKRmice161

anddownregulated inBALB/cmice(1-IPPLR<0.05),65weresuccessfullymatchedtogene162

IDs in DAVID. The gene upregulated in colitis-susceptible AKR mice with the largest163

differentialexpressioncompared toBALB/cmicewas the receptor foradvancedglycation164

end-products(Rage)(Log2foldchange=2.0718;1-IPPLR=0.003)(Figure2B;Supplementary165

data).UpregulationofRageinsusceptiblemicewasconfirmedbyqPCRofproximalcolonat166

24hoursPIinanindependentexperiment(P<0.001,Mann-WhitneyUtest,Figure2C).167


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DifferentialexpressionoftranscriptionfactorswasanalysedusingTIGERiforMATLAB[10].168

Themostnotablechangeintranscriptionfactorgeneexpressionwasthedownregulationof169

FoxO4 (Forkhead boxO4) followingT.muris infection in AKRmice (Supplementary data).170

FOXO4occursdownstreamof theRAGEactivationsignallingcascadeandserves to inhibit171

DNA binding and transcriptional activity of NF-κB (nuclear factor kappa-B), preventing172

inflammation[11].FoxO4isalsodownregulatedinthecolonicepithelialcellsofIBDpatients173

[11]. TheupregulationofproinflammatoryRageanddownregulationofanti-inflammatory174

FoxO4fromtheRAGEsignallingpathwayprovidecompellingevidencefortherelevanceof175

RAGEactivationincolitissusceptibilityinAKRmiceduringT.murisinfection.176

IdentifyingthecellularsourceofRAGE177

Over90%ofCD326+(EpCAM)epithelialcellsexpressedRAGE(Figure3A)andtheyfellinto178

twodistinctgroups,expressingeitherlow(RAGElo)orhigh(RAGEhi)levelsofRAGE.Innaïve179

mice, thetotalproportionofCD326+epithelialcells thatexpressedRAGEwassignificantly180

higherincolitis-susceptibleAKRmice(P<0.01,ANOVA).TheproportionofRAGElotoRAGEhi181

cellswassimilar inbothnaïveAKRandBALB/cmice,buttherewassignificantdrop inthe182

proportionofRAGEhiepithelialcellsobserved2and7daysPI (P<0.0001,ANOVA), inboth183

AKRandBALB/cmice(Figure3B).184

ImmunohistochemistrywasusedtoconfirmexpressionofRAGEintissue(Figure3C).Akinto185

the flow cytometry data, we saw high expression of RAGE throughout the colonic186

epithelium.Concurringwiththeflowcytometrydata,therewasonlyminimalfluorescence187

seen in the immune lamina propria cells indicating that epithelial cells indeed express188

greateramountsofmembrane-boundRAGEthanimmunecells.Thisdatasuggestthatthe189

epithelialcellsarelikelytobethesourceoftheobservedincreaseinRagemRNA.Therewas190

areductioninintensityofRAGEstainingat7daysPIbyimmunohistochemistry(Figure3C)191


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compared tonaïvemice,which also correlatedwith themeasured shift in proportionsof192

epithelialcellsfromRAGEhitoRAGElocellsmeasuredbyflowcytometry.193

IsRAGEdifferentiallycleavedincolitis-susceptibleandcolitis-resistantmice?194

RAGE may be internalised after ligand binding, or released as soluble RAGE (sRAGE) via195

enzymaticcleavagebyADAM10orMMP9[12,13].ToinvestigatewhetherRAGEwasbeing196

cleavedweassessedsRAGElevelsinserumandthefaecesbyELISA.SerumsRAGElevelsin197

susceptible mice remained constant throughout the experiment. Resistant mice had198

significantly higher serum sRAGE than susceptiblemice both prior to infection and at 24199

hours PI (P<0.001, ANOVA; Figure 3D). sRAGEwas also detectable in faeces,where both200

resistant and susceptiblemice had very low levels of sRAGE prior to infection. Over the201

courseofinfection,faecalsRAGEinBALB/cmiceincreasedtosignificantlyhigherlevelsthan202

susceptiblemiceat24hoursand21daysPI(P<0.01,ANOVA;Figure3E).FaecalsRAGEwas203

notdetectedatalluptoD21insusceptiblemice.204

CorrelationofserumandfaecalsRAGElevelstocolitisscoreshighlightsthechanginglevels205

of sRAGE in BALB/cmice during the course of T. muris infection relative to pathological206

changesinthecolon(Figure3F-G).TheincreasedcirculatingserumsRAGEatD0inBALB/c207

mice,where colitis scores are lowest, drops as colitis increases at D1 andD21 (R2=0.90).208

Faecal sRAGE in the BALB/c mice increases relative to colitis scores (R2=0.99). However,209

sRAGE levels in susceptiblemicedidnot changeduring thecourseof infection relative to210

increasingcolitisscoresfromD0toD21post-infection(serumR2=0.99,faecalR2=0.73).211

We then investigated levels of the RAGE ligand S100A8 (one part of the heterodimeric212

calprotectinprotein,currentlyusedasaclinicalbiomarkerfor IBD) inserumandfaecesas213

an indicator of whether sRAGE might be quenching the proinflammatory effects of214

circulating RAGE ligands by acting as a decoy receptor. Serum and faecal S100A8 did215


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increase slightly during the course of infection in both AKR and BALB/c mice, but no216

statisticaldifferenceswereobservedbetweenthetwostrainsandtherewashighvariability217

betweenmice.At21daysPIBALB/cmicehadgreaterlevelsofserumS100A8thanAKRmice218

(not significant, ANOVA; Supplementary data). Faecal S100A8 remainedbroadly similar in219

naïveandT.muris infectedmiceofbothAKRandBALB/c strains.Aswith serumS100A8,220

faecalS100A8wasslightly raised inBALB/cmiceat21daysPIcomparedtoAKRmicebut221

thiswasnotsignificant(ANOVA;(Supplementarydata)).Inadditiontobeinghighlyvariable,222

S100A8correlatedpoorlywithcolitisscores(Supplementarydata).223

sRAGEisdifferentiallyexpressedinIBD224

As the differences in sRAGEweremost apparent and consistent in faeceswe focusedon225

analysis of faecal specimens from healthy volunteers and patientswith IBD or suspected226

IBD. sRAGEwas not detected in the faecal samples of healthy volunteers. In contrast, s-227

RAGEwasdetectable in thepatient cohort (Figure4A). Thehighest levels of sRAGEwere228

seen in patients with IBD excluded (largely IBS ascribed) and IBD in remission, although229

remission patients were more variable. Patients with active IBD characterised by severe230

inflammationhadlowlevelsofsRAGEandincreasedcalprotectin.231

WethentransformedthefaecalRAGEandcalprotectinELISAdataintopresent(1)orabsent232

(0),where protein is scored as present if ≥ 3*SD abovebaseline.Whenpatient datawas233

stratifiedintogroups(activeIBD,IBDinremission,IBDexcluded(IBS)andhealthycontrols)234

theratioofRAGEtocalprotectinclearlyidentifiedhealthycontrols(0,0)andactiveIBD(0,235

1) from IBS and IBD in remission (Figure 4B). In healthy individuals,we sawno sRAGEor236

calprotectinsignalinanysubject.InactiveIBDwehadaconsistentpatternofCalprotectin237

present,butnosRAGE.Forpatientswhosediseaseisresolvingwesawamorecomplicated238


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picture,wherewehad a subset of patients undergoing routine test thatwere expressing239

bothsRAGEandCalprotectin. 240


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Discussion241

Animalmodelsarecrucialforexaminingthecausativeeventsthatleadtodiseasessuchas242

chronic colitis. Genetic factors influence the likelihood of developing colitis, but it is243

impossible to continually monitor individuals with potential genetically susceptibility in244

order to identify the pathways that drive the development of chronic intestinal245

inflammation.Models that accurately simulate preclinical changes in the gut allow us to246

interrogatethepathwaysleadingtochroniccolitis,priortothedevelopmentofthecomplex247

inflammatoryenvironmentwhendisease isestablished.Levisonetal. [14]havepreviously248

shownthatT.muris infectioninAKRmicecausescolitisthatcorrelatesphenotypicallyand249

transcriptionallywiththeprofileofhumanCD.Here,weprovidenovelevidencereinforcing250

the use of the T. muris infection as a model for the discovery of preclinical intestinal251

inflammationmarkersthattranslateintohumanIBDpatients.252

In line with previous T. muris infection studies, we observed altered dynamics of the253

immune response between colitis resistant versus susceptible mice, characterised by an254

early influxofDCs [5].Wethen identifiedupregulationofRageasapotential indicatorof255

colitissusceptibilityinmice.RAGEactivityhasalreadybeenlinkedtoactiveIBD[15]aswell256

asotherinflammatorydiseasesincludingdiabetes,Alzheimer’s,airwayinflammation,cancer257

and haemorrhagic shock [16]. Additionally, several RAGE ligands have been identified as258

associatedwiththe inflammation in IBD, includingcalprotectin,EN-RAGEandHMGB1[17-259

19].Thus,ourdatafromthemousemodelshowsaclearlinktoknownpathologyinIBD.Itis260

important to validate results from mouse model to human disease and therefore we261

conducted a small validation study to assess faecal sRAGE. Faecal sRAGE was readily262

detected in patient’s faecal samples. Akin to the mouse data we saw lower sRAGE in263

patients with active chronic inflammation. Surprisingly, symptomatic patients with IBD264


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excluded on a basis of normal FCP and/or colonoscopy had higher levels of sRAGE than265

healthy volunteers. Largely, IBSwas clinically ascribed but thatwas not based on formal266

diagnostic criteria, and other diagnoses such as bile acid diarrhoea ormicroscopic colitis267

werenot formally excluded. The sample sizewas small andmoreprospective studies are268

neededtoconfirmthispreliminaryobservation.PatientswhoseIBDwasreportedtobe in269

remissionhadvariablelevelsofsRAGE.ItistemptingtospeculatethatlowerlevelsofsRAGE270

are associatedwith a risk of subsequent flare of inflammation but aswe only had single271

samplesfromthepatients,wecannotassessthis,howeveritwouldbeinterestingtotrack272

sRAGEovertimeinaprospectivestudyofpatientswithIBD.273

RAGEhasbeendescribedonseveralimmunecellswith,forexample,neutrophilsidentified274

asexpressing largeamountsofRAGE[20].Ourflowcytometryandimmunohistochemistry275

analysisofRAGEexpressioninmultiplecelltypespresentinandaroundthelaminapropria276

andcryptsofthecolon,however,didnotsuggestthatimmunecellswerethemainsources277

of cellularRAGE.Ourdata in fact showed thatamajor cellular sourceofRAGE in thegut278

were the gut epithelial cells. Previous studies have shown that epithelial cells not only279

express RAGE, but also upregulate RAGE expression during colonic inflammation [15].280

Changesweobserved in the levelsofRAGEexpressionsuggest that it is theepithelialcell281

response to T. muris infection that informs subsequent susceptibility to chronic282

inflammation.ThereductionintheamountofRAGEpresentatthecellmembranewesaw283

by flowcytometryandby immunohistochemistry immediately followingT.muris infection284

couldbecausedbyeither internalisationofactivatedRAGE-ligandcomplexesorADAM10-285

mediatedsheddingtoproducesRAGE[21,22].SplicevariantsofRagemayalsoresult ina286

truncatedRAGEmoleculeoramodifiedandactivelysecreteddecoyreceptor[23,24].The287

process by which epithelial cells may undergo RAGE shedding represents an important288


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distinction in the course of gut immunity and homeostasis, and may be an essential289

component in dictating whether inflammation becomes chronic or resolves. Further290

investigation into the extent towhich splice variants, internalisation or sheddase activity291

form the mechanism for changes in sRAGE in both the Trichuris model and in human292

patientscouldprovidefurtherinsightfortheroleoftheRAGEpathwayincolitis.293

Activation of RAGE can have several outcomes including immune cell migration and294

transcriptionofpro-inflammatorycytokines.RAGE-mediatedleukocytemigrationviaCD11b295

(Mac-1)isinvolvedinmigrationofimmunecellstothesiteofinjuryandhomingofDCsto296

thelymphnodes[15,25].However,whethertheupregulationofRageiscrucialtofacilitate297

the early DC migration in the T. muris model remains unclear. While we observed298

differencesinDCmigrationasearlyasday1PI,wesawnodifferencesbetweencellsurface299

RAGE expression between AKR and BALB/c mice that might account for the altered300

dynamicsofDCrecruitment.Similarly,RAGEhasbeenlinkedtoneutrophilrecruitmentbut301

weonlyobservedmodestneutrophil infiltration in the first24hoursPIandnodifference302

betweencolitis-susceptibleorcolitis-resistantmice.Despiteminimaldifferencesinimmune303

cell presence during the early stages of T.muris infection, prolonged activation of RAGE304

resultsinactivationofinflammatorysignallingmoleculesincludingNF-κBandMAPkinases305

[16].Consequently,anenvironmentwhereRAGEligandssuchasHMGB1,andS100proteins306

are continually present results in perpetual NF-κB activation and subsequent chronic307

inflammatoryconditions.308

WeobservedstrikingdifferencesinthelevelsoffaecalandsystemicsRAGEbetweencolitis-309

resistantandsusceptiblemice,withBALB/cmicerapidlyproducingsRAGEinresponsetoT.310

murisinfection.sRAGEeffectivelyactsasadecoyreceptorforRAGEasitcanstillbindtothe311

samedamageinducedligandsasmembraneboundRAGEbut,asitlacksacytoplasmictail,it312


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cannot initiate the pro-inflammatory signalling cascade [26]. Thus, higher levels of sRAGE313

mightbeexpected toblock inflammation and indeed reduced levels of sRAGEhavebeen314

found in mice with chronic inflammation as well as patients suffering from chronic315

inflammatory diseases [27]. The reduction in epithelial RAGE expression followed by316

increases in circulating sRAGE in colitis-resistantmice suggests sheddingof RAGE to form317

sRAGEasaprotectivefeedbackprocessagainstthedevelopmentofchronic inflammation.318

Colitis-susceptibleAKRmiceshowthesamereductioninepithelialRAGEexpressionbutdid319

notproducesRAGEinthesamequantities,suggestinginternalisationandactivationofRAGE320

andinitiationofsubsequentproinflammatorypathwaysafterT.murisinfection.Indeed,itis321

known thatT.muris excretory/secretory (E/S) products induceNF-κB signalling in colonic322

epithelial cells shortly after infection and the susceptible immune response toT.muris is323

associated with expression of the T helper (TH) 1 cytokines interferon (IFN)-γ, tumour324

necrosisfactor(TNF)-αandIL-12[28].Thisresponseparallelsthecytokinesexpressedafter325

RAGEactivation,whichincludeTH1andTH17cytokinesTNF-α,IL-1α,IL-6,IL-8andIL-12[16].326

DiagnosisofIBDusuallyinvolvesanassessmentofclinicalhistoryandphysicalexamination,327

with endoscopy and histology considered to be the gold standard tools [29]. Accurately328

assessingdiseaseactivityremainsdependentoncolonoscopyand/orsmallbowel imaging.329

The invasivenessofcurrentdiagnosticmethods isnot idealandrecentworkhasaimedto330

identify serum or faecal biomarkers that can reliably identify active disease [30]. The331

number of potential IBD biomarkers is high, but there remains a lack of reliable and332

reproduciblebiomarkersforuseinclinicalpractice[31].Efficacyofcurrenttherapiesisalso333

variable,withrisksofsometimesserioussideeffects,especially infectionmeaningthere is334

considerable interest fornewbiomarkersandnewtherapeutics [32].Calprotectinentered335

clinical practice as an IBD biomarker to aid clinical diagnosis non-invasively, but336


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measurementsoffaecalcalprotectinarevariableandthereis littleagreementaboutwhat337

shouldbeconsideredanormalbaselinelevelinhealthypatients[33].Indeed,theconceptof338

a simple normal cut off is impossible to entertain given the enormous heterogeneity in339

faecalwatercontent,matrixcomposition,transittime,siteandextentofinflammationand340

the contact of faecal component sampled with the mucosa; composite measures are341

essential. Calprotectin is a product of tissue damage and binds to RAGE to promote342

inflammation,butthisactionwillbereducedinthepresenceofthedecoyreceptorsRAGE.343

OurpreliminaryobservationofalterationsinsRAGEexpressioninmouseandhumandisease344

suggests theremay bemerit in looking at both calprotectin and sRAGE to better predict345

whether calprotectin and other RAGE ligands are indeed able to drive pro-inflammatory346

signals.Thismayimprovethereliabilityofcalprotectinasabiomarker.347

Our pilot clinical study successfully validated the use of the T. muris infection model as348

highly translatable to human IBD states. TheT.murismodel is a useful tool in dissecting349

early pathways that are involved in the onset of colitis. By using a mouse model and350

focusing on early initiating events in the development of colitis, we have identified a351

potential role forRAGE inmediating thedevelopmentof inflammation. Furthermore, our352

observationofhighlevelsofsRAGEinacuteresolvinginflammationsuggestedtheremaybe353

utility inmonitoring of sRAGE tomonitor IBD. However, a larger clinical study would be354

requiredtoinvestigatethisfurther.355

Acknowledgements356

Funding for this researchwasprovidedby theEPSRC,EpistemLtd.and theBBSRC impact357

accelerator fund. NHS Salford Trust provided the human clinical samples for this project,358

andwearegratefultoallclinicalstaff,patientsandhealthyvolunteerswhotookpartinthe359

trial.360


https://doi.org/10.1101/719310

The Bioimaging Facilitymicroscopes used in this study were purchasedwith grants from361

BBSRC,Wellcome and the University ofManchester Strategic Fund. Special thanks go to362

RogerMeadowsfrom the Bioimaging Facility for his helpwith themicroscopy and image363

acquisition. 364


https://doi.org/10.1101/719310

19

References365

1. Baumgart, D.C. and S.R. Carding, Inflammatory bowel disease: cause and366immunobiology.Lancet,2007.369(9573):p.1627-40.367

2. Kaplan,G.G.,Theglobalburdenof IBD: from2015 to2025.NatRevGastroenterol368Hepatol,2015.12(12):p.720-7.369

3. Liverani, E., et al., How to predict clinical relapse in inflammatory bowel disease370patients.WorldJGastroenterol,2016.22(3):p.1017-33.371

4. Levison, S.E., et al.,Genetic analysis of the Trichurismuris-inducedmodel of colitis372revealsQTLoverlapandanovelgenecluster forestablishingcolonic inflammation.373BMCGenomics,2013.14:p.127.374

5. Cruickshank, S.M., et al., Rapid dendritic cell mobilization to the large intestinal375epithelium is associated with resistance to Trichuris muris infection. Journal of376Immunology,2009.182(5):p.3055-62.377

6. Bowcutt, R., et al.,A role for the pattern recognition receptor Nod2 in promoting378recruitment of CD103+ dendritic cells to the colon in response to Trichuris muris379infection.MucosalImmunol,2014.7(5):p.1094-105.380

7. Foell,D.,H.Wittkowski, and J.Roth,Monitoringdiseaseactivityby stoolanalyses:381fromoccultbloodtomolecularmarkersofintestinalinflammationanddamage.Gut,3822009.58(6):p.859-68.383

8. Wakelin, D.,Acquired immunity to Trichurismuris in the albino laboratorymouse.384Parasitology,1967.57(3):p.515-24.385

9. Else,K.J.,etal.,Theinfluenceofgenesmappingwithinthemajorhistocompatibility386complexonresistancetoTrichurismurisinfectionsinmice.Parasitology,1990.101Pt3871:p.61-7.388

10. Han,N.,H.A.Noyes,andA.Brass,TIGERi:modelingandvisualizingtheresponsesto389perturbationofa transcription factornetwork.BMCBioinformatics,2017.18(Suppl3907):p.260.391

11. Zhou,W.,etal.,FoxO4 inhibitsNF-kappaBandprotectsmiceagainstcolonic injury392andinflammation.Gastroenterology,2009.137(4):p.1403-14.393

12. Curran,C.S.andP.J.Bertics,HumaneosinophilsexpressRAGE,produceRAGEligands,394exhibit PKC-delta phosphorylation and enhanced viability in response to the RAGE395ligand,S100B.IntImmunol,2011.23(12):p.713-28.396

13. Zhang,L.,etal.,Receptorforadvancedglycationendproductsissubjectedtoprotein397ectodomain sheddingbymetalloproteinases. JBiolChem,2008.283(51):p.35507-39816.399

14. Levison,S.E.,etal.,Colonictranscriptionalprofilinginresistanceandsusceptibilityto400trichuriasis: phenotyping a chronic colitis and lessons for iatrogenic helminthosis.401InflammBowelDis,2010.16(12):p.:2065-79.402

15. Body-Malapel, M., et al., The RAGE signaling pathway is involved in intestinal403inflammationandrepresentsapromisingtherapeutictargetforInflammatoryBowel404Diseases.MucosalImmunol,2019.12(2):p.468-478.405

16. Sparvero, L.J., et al., RAGE (receptor for advanced glycation endproducts), RAGE406ligands,andtheirroleincancerandinflammation.JournalofTranslationalMedicine,4072009.7.408


https://doi.org/10.1101/719310

17. Whitehead, S.J., et al., Effect of faecal calprotectin assay variability on the409managementofinflammatoryboweldiseaseandpotentialroleoffaecalS100A12.J410ClinPathol,2017.70(12):p.1049-1056.411

18. Chen,X.,etal.,HMGB1exacerbatesexperimentalmousecolitisbyenhancinginnate412lymphoid cells 3 inflammatory responses via promoted IL-23 production. Innate413Immun,2016.22(8):p.696-705.414

19. Aranda,C.J.,etal.,Calprotectinprotectsagainstexperimentalcolonic inflammation415inmice.BrJPharmacol,2018.175(19):p.3797-3812.416

20. Huebener, P., et al., The HMGB1/RAGE axis triggers neutrophil-mediated injury417amplificationfollowingnecrosis.JClinInvest,2019.130:p.1802.418

21. Sevillano, N., et al., Internalization of the receptor for advanced glycation end419products (RAGE) is required to mediate intracellular responses. J Biochem, 2009.420145(1):p.21-30.421

22. Yang,W.S.,etal.,EctodomainSheddingofRAGEandTLR4asaNegativeFeedback422Regulation in High-Mobility Group Box 1-Activated Aortic Endothelial Cells. Cell423PhysiolBiochem,2018.51(4):p.1632-1644.424

23. Hudson,B.I.,etal., Identification,classification,andexpressionofRAGEgenesplice425variants.FASEBJ,2008.22(5):p.1572-80.426

24. Jules, J., D.Maiguel, andB.I.Hudson,Alternative splicing of theRAGE cytoplasmic427domainregulatescellsignalingandfunction.PLoSOne,2013.8(11):p.e78267.428

25. Manfredi,A.A.,etal.,MaturingdendriticcellsdependonRAGEforinvivohomingto429lymphnodes.JImmunol,2008.180(4):p.2270-5.430

26. Basta,G.,etal.,Circulatingsolublereceptor foradvancedglycationendproducts is431inversely associated with glycemic control and S100A12 protein. J Clin Endocrinol432Metab,2006.91(11):p.4628-34.433

27. Maillard-Lefebvre,H.,etal.,Solublereceptorforadvancedglycationendproducts:a434new biomarker in diagnosis and prognosis of chronic inflammatory diseases.435Rheumatology,2009.48(10):p.1190-1196.436

28. deSchoolmeester,M.L., H.Manku, and K.J. Else, The innate immune responses of437colonicepithelialcellstoTrichurismurisaresimilar inmousestrainsthatdevelopa438type1ortype2adaptiveimmuneresponse.InfectImmun,2006.74(11):p.6280-6.439

29. Halfvarson, J.,Geneticepidemiologyof inflammatoryboweldisease,early twinand440familystudies,inMoleculargeneticsofinflammatoryboweldisease,M.D'amatoand441J.D.Rioux,Editors.2013,Springer:NewYork.p.23-43.442

30. Lehmann, F.S., E. Burri, and C. Beglinger, The role and utility of faecalmarkers in443inflammatoryboweldisease.TherapAdvGastroenterol,2015.8(1):p.23-36.444

31. Stevens, T.W., et al., Systematic review: predictive biomarkers of therapeutic445responseininflammatoryboweldisease-personalisedmedicineinitsinfancy.Aliment446PharmacolTher,2018.48(11-12):p.1213-1231.447

32. Nanau,R.M.,L.E.Cohen,andM.G.Neuman,Riskofinfectionsofbiologicaltherapies448withaccentoninflammatoryboweldisease.JPharmPharmSci,2014.17(4):p.485-449531.450

33. Dhaliwal,A.,etal.,Utilityoffaecalcalprotectinininflammatoryboweldisease(IBD):451whatcut-offsshouldweapply?FrontlineGastroenterol,2015.6(1):p.14-19. 452


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21

Figurelegends453

Figure1:Colitis-susceptibleAKRmiceshowdelayedexpulsionofTrichurismuriswormsat454

21daysandincreasedevidenceofcolitisat31dayspostinfection.(A)Meanwormburden455

(±SD)at21dayspostinfection(PI).(B)Cumulativecolitisscore(0-20)basedonthegrading456

ofhistologicalchangesincludingcryptelongation(score0-4),depletionofgobletcells(score457

0-4), thickness of muscle wall (score 0-4), inflammatory cell infiltration (score 0-4) and458

destructionofarchitecture(score0or3-4).(C)Representativeimagesofhaematoxylinand459

eosinstainedproximalcolonsectionsfromnaïvemiceandat31daysPI;notethehighlevels460

of immunecell infiltrationand lossofgobletcells inthecolonictissuesofAKRmiceat31461

dayspost-infection.Bar=50μm.n=3-5micepertimepoint.AnalysisbyMann-WhitneyUtest462

ortwo-wayANOVAfollowedbySidak’smultiplecomparisonstestwhereappropriate.(D-G)463

Dendriticcell (CD45+MHCII+CD11c+F4/80-CD103+/-CD11b+/-),macrophage(CD45+MHCII+464

F4/80+ CD11c+/-), inflammatory monocyte (CD45+ Ly6G+ CD11b+ CD115+) and neutrophil465

(CD45+Ly6G+CD11b+CD115-)populationsasproportionofCD45+cells(±SEM)innaïvemice466

andduringT.muris challenge.n=3miceper timepoint.Analysisby two-wayANOVAwith467

Sidak’smultiplecomparisonsposthoctest.***P<0.001,**P<0.01,*P<0.05. 468

469

Figure2:Geneexpressionchangesinproximalcolon24hourspost-infectionwithTrichuris470

muris.(A)GenesmostsignificantlyupregulatedinAKRmiceanddownregulatedinBALB/c471

mice(red)ordownregulatedinAKRmiceandupregulatedinBALB/cmice(blue)following472

24hourTrichurismurisinfection.(B)SchematicofthestructureofRAGE,showingactivating473

ligands,downstreamNF-κBactivationandformationofsolubleRAGE(sRAGE)byADAM10474

orMMP9cleavage.(C)mRNAexpressionofRAGEintheproximalcolonatD1post-infection475

asmeasuredbyqPCR.DatageneratedusingAffymetrixMouse4302.0microarraysanalysed476


https://doi.org/10.1101/719310

usingthepumaandTIGERi(TFAillustratorforglobalexplanationofregulatoryinteractions)477

packages for Bioconductor. n=4-5 mice per group. Analysis by Mann-Whitney U-test.478

**P<0.01. 479

480

Figure3:Receptorforadvancedglycationend-products(RAGE)expressioninthecolonof481

Trichurismuris infectedAKRandBALB/cmice (aged6-8weeks). (A) ProportionofRAGE482

expressing epithelial cells (CD326+, ±SEM) during early Trichuris muris infection. (B)483

ProportionofcolonicepithelialcellsexpressinghighlevelsofRAGEisreducedshortlyafter484

infection, as measured by flow cytometry. (C) Representative images of colon sections485

stained for RAGE (FITC; green) and nuclei (DAPI; blue) in naïvemice and at 7 days post-486

infection(Bar=100μm; insetbar=22μm). (D-E)sRAGEpresent inserumorfaecesduring487

TrichurismurisinfectionasmeasuredbyELISA.(F-G)CorrelationofserumandfaecalsRAGE488

versuscolitisscoreat0,1and21dayspost-infection.n=3-5micepertimepoint.Analysisby489

linearregression,two-wayANOVAwithSidak’sposthoctest.**P<0.01****P<0.001. 490

491

Figure 4: Soluble receptor for advanced glycation end-products (sRAGE) is detectable in492

thefaecesandserumofIBDpatients.(A)ScatterplotofsRAGE(pg/ml)versuscalprotectin493

(μg/ml)presentinthefaecesofpatientswithactiveIBD,IBDinremission,IBDexcluded(IBS)494

compared to healthy controls. (B) Relative levels of faecal sRAGE versus calprotectin in495

human IBD/IBS or healthy controls. Datawere transformed to arbitrary units (AU)where496

samplesgreaterthan3SDfrombaseline=1(present),otherwisetheyscored0(absent).497


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AKR BALB/c

D0

D31

A

B

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D E

F G

DCs

0 1 7 14 21 310

5

10

15

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Time post-infection

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0 1 7 14 21 3105

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% o

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Inflammatory monocytes

0 1 7 14 21 310

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**

Time post-infection

% o

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Neutrophils

0 1 7 14 21 310

10

20

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% o

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45+

cells

AKRBALB/c *** *** ***

Figure 1


https://doi.org/10.1101/719310

Rag

eG

m35

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dhr4

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Figure 2


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0 2 4 6 80

20000

40000

60000

Colitis Score

Ser

um s

RA

GE

(pg/

ml)

Serum sRAGE vs colitis score

AKR BALB/c

D0

D7

A

B

C

D

0 2 4 6 80

5000

10000

15000

Colitis Score

Faec

al s

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GE

(pg/

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Faecal sRAGE vs colitis score

AKR

BALB/c

E

F G

0 2 70

20

40

60

80

100

Timepoint post-infection

% o

f CD3

26+

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Epithelial cell RAGE expression

AKRBALB/c

**

0 2 70

20

40

60

80

100

Timepoint post-infection

% o

f RAG

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Figure 3


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A B

sRAGE Calprotectin0.0

0.5

1.0

Aver

age

AU

Active IBDIBD remission IBD excludedHealthy

0 200 400 6000

500

1000

1500

sRAGE pg/ml

Calp

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μg/

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Active IBDIBD remissionIBD excludedHealthy

Figure 4


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differential expression of soluble receptor for …...2019/08/05 · 21 s. m. cruickshank, av hill...

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