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1NOD-likereceptorsignalingandinflammasome-relatedpathwaysarehighlightedinpsoriaticepidermisMariH.
Tervaniemi1,2,ShintaroKatayama3,4,TiinaSkoog3,H.
AnnikaSiitonen1,2,JyrkiVuola5,KristoNuutila6,RaijaSormunen7,AnnaJohnsson8,StenLinnarsson8,SariSuomela9,EskoKankuri6,JuhaKere1,2,3,4&OutiElomaa1,2Psoriaticskindiffersdistinctlyfromnormalskinbyitsthickenedepidermis.
Mostgeneexpressioncomparisonsutilizefull-thicknessbiopsies,withsubstantialamountofdermis.
Weassayedthetranscriptomesofnormal,lesional,andnon-lesionalpsoriaticepidermis,sampledassplit-thicknessskingrafts,with5′-endRNAsequencing.
WefoundthatpsoriaticepidermiscontainsmoremRNApertotalRNAthancontrols,andtookthisintoaccountinthebioinformaticanalysis.
Theapproachhighlightedinnateimmunity-relatedpathwaysinpsoriasis,includingNOD-likereceptor(NLR)signalingandinflammasomeactivation.
WedemonstratedthattheNLRsignalinggenesNOD2,PYCARD,CARD6,andIFI16areupregulatedinpsoriaticepidermis,andstrengthenedthesefindingsbyproteinexpression.
Interestingly,PYCARD,thekeycomponentoftheinflammasome,showedanalteredexpressionpatterninthelesionalepidermis.
Theprofilingofnon-lesionalskinhighlightedPSORS4andmitochondriallyencodedtranscripts,suggestingthattheirgeneexpressionisalteredalreadybeforethedevelopmentoflesions.
Ourdatasuggestthatallcomponentsneededfortheactiveinflammasomearepresentinthekeratinocytesofpsoriaticskin.
Thecharacterizationofinflammasomepathwaysprovidesfurtheropportunitiesfortherapy.
Complementingprevioustranscriptomestudies,ourapproachgivesdeeperinsightintothegeneregulationinpsoriaticepidermis.
Psoriaticskinischaracterizedbythehyperproliferationandabnormaldifferentiationofkeratinocytesandinfil-trationofinflammatorycells.
Componentsofthecornifiedenvelope,theoutermostlayerofepidermis,arepre-maturelysynthesizedinthespinouslayerthatisthickeranddisorganizedinpsoriasis.
Theinflammatoryinfiltrateconsistsmainlyofdendriticcells,macrophages,andTcellsinthedermisandneutrophilswithsomeTcellsintheepidermis1,2.
Geneexpressionintheepidermisisdramaticallyalteredduringthepathogenesisofpsoriasis.
Forexample,severalgenesoftheepidermaldifferentiationcomplex(EDC)region(1q21)areupregulatedinthepsoriaticlesions.
Theseincludegenesthatplayaroleinthegenerationandmaintenanceoftheepidermis:cornifiedenvelopeprecursors(e.
g.
,smallproline-richproteins;SPRRs),latecornifiedenvelopeproteins(LCEs),andsignalingproteins(e.
g.
,sS100calcium-bindingproteins;S100s).
TheEDCregionalsocontainsthepsoriasissusceptibilitylocus4(PSORS4)3,4.
Theregulationofinflammationinthepsoriaticskinrequirescross-talkbetweenthekeratinocytesandtheimmunecells.
Keratinocytesproduceseveralanti-microbialpeptidesandproteins(e.
g.
,LL37,β-defensis,andinterferon-γ)thatattractimmunecellsandshapetheirfunctions.
Theyalsorespondtoimmunecell-derivedcytokines,suchasinterferons,interleukin-17,andtheinterleukin-20familyofcytokines,andinturnproduce1FolkhlsanInstituteofGenetics,Helsinki,Finland.
2DepartmentofMedicalandClinicalGenetics,MedicumandResearchProgramsUnit,MolecularNeurology,UniversityofHelsinki,Helsinki,Finland.
3DepartmentofBiosciencesandNutrition,KarolinskaInstitutet,Huddinge,Sweden.
4ScienceforLifeLaboratory,Solna,Sweden.
5HelsinkiBurnCenter,DepartmentofPlasticSurgery,UniversityofHelsinkiandHelsinkiUniversityHospital,Helsinki,Finland.
6DepartmentofPharmacology,Medicum,UniversityofHelsinki,Helsinki,Finland.
7BiocenterOulu,DepartmentofPathology,UniversityofOulu,Oulu,Finland.
8DepartmentofMedicalBiochemistryandBiophysics,KarolinskaInstitutet,Stockholm,Sweden.
9DepartmentofDermatology,UniversityofHelsinkiandHelsinkiUniversityHospital,Helsinki,Finland.
CorrespondenceandrequestsformaterialsshouldbeaddressedtoJ.
K.
(email:juha.
kere@ki.
se)received:15October2015accepted:19February2016Published:15March2016OPEN2proinflammatorycytokines(interleukin-1andTNF-α)1.
Inaddition,theyrecognizepathogensandendogenouscellularstresssignalsviapatternrecognitionreceptors(PRRs):e.
g.
,NOD-like(NLR)andRIG-likereceptors(RLR),andthereforemediateimmuneresponses5.
Severalmicroarraystudiesofthepsoriaticlesionalskinhaverevealedalargenumberofdifferentiallyexpressedgenes(DEGs)incomparisonswithcontrolandnon-lesionalskin6,7.
Recently,RNAsequencing(RNAseq)hasprovidedanewalternativetomicroarrays;sofar,twoRNAseqstudiesonpsoriasishavebeenpublished8,9.
However,mostofthepreviouspsoriasistranscriptomeanalysesusedfull-thicknessskinsamples.
Hereweaimedtofocusonepidermalchangesinpsoriasis.
WeutilizedRNA5′-endtargetedsequencingforsplit-thicknessskingrafts(SG)thatarecomposedoftheepidermis(SupplementaryFigureS1)andincludemuchlessdermisthanthefull-thicknesssamples.
Wecollectedsamplesfromthepsoriaticlesionalandnon-lesionalskinfrompatientsandhealthyskinfromcontrols.
Wealsoemployeddatanormalizationwithsyntheticspike-inRNA,whichenablesmoreaccuratecomparisonofgeneexpressioninheterogeneoussampleswheretotalmRNAlevelspercellmaybehighlydifferent10–12.
OurresultsshowthepowerofRNAseqoverthemicroarraystudies,providingamorecomprehensiveviewofalteredsignalingpathwaysbothinnon-lesionalandlesionalpsoriaticskin.
ThesensitivityoftheRNAseqmethod,togetherwiththeskingraftsamples,allowsamorein-depthiden-tificationofalteredcomponentsineachpathway,makingitpossibletogetabetteroverallunderstandingaboutaffectedpathwaysandnetworksinpsoriaticepidermis.
ResultsRNAseqofpsoriaticskingraftsidentifiedasubstantialnumberofdifferentiallyexpressedtran-scripts.
TotalRNAsamplesof10ng,extractedfromninecontrol(C),fivepsoriaticnon-lesional(PN),andsixlesional(PL)SGs(Table1,SupplementaryTableS1),weresubjectedto5′-endRNAseq10.
Fordifferentialexpressionanalysis,weemployedtheSAMstrtstatisticalpackage,whichisbasedonsyntheticspike-inRNAnormalizationofdata11,12.
Weestimatedthedissimilaritybetweenthesamplesbyprincipalcomponentanalysis(PCA)(Fig.
1a),whichillustratessignificantoverlapofthehealthyskinsamples(PNandC)butseparationfromthelesionalsamples.
Somenon-lesionalsamplesclusteredbetweenthecontrolandlesionalsamples,suggestingchangesalreadyinnon-lesionalskin.
Thegroup-wise(GW)comparisonsoftranscriptsbetweenlesionandcontrol(PLvsC)ornon-lesion(PLvsPN)revealed2436and3541upregulatedand2550and494downregulateddifferentiallyexpressedtranscripts(DETs),respectively(SupplementaryTableS2a–f)(FoldChange(FC)>1.
5and1*108).
AlsoseveralothercomponentsinvolvedintheNLRsignalingwereidentifiable,suchasNLRP10,NLRX1,CASP1,CASP8,andPYCARD(ASC).
MostofthesegeneswereidentifiableinthePLvsCcomparisonaswell.
Furthermore,thereceptorsofthecytosolicDNAsensingandRLRpathways;DNA-bindingreceptorsAIM2andIFI16andRNAhelicaseproteinsIFIH1andDDX58(RIG-I),wereinducedinthelesionalsamples.
AlsoseveralotherRLR-relatedtranscriptswereupregulated,includingISG15andCYLD(Table2).
Weshowedrecently,byqPCR,thatourRNAseqprotocolenablestheaccuratequantitationofgeneexpres-sioninskin12.
PatientsamplesofthepresentstudywereprocessedandanalyzedatthesametimeassamplesofFigure1.
Expressionprofilingoflesionalandnon-lesionalpsoriaticandcontrolskinbyRNAseq.
(a)Principalcomponentanalysisdemonstratesclusteringofthespike-innormalizedsamplegroupsPL(red),PN(blue),andC(green).
Percentagesarecontributionratios.
Thethreelibrarieshavedifferentsymbols;L247andL248indicateRun1andL293Run2(descriptioninSupplementaryTableS1)(b)VenndiagramofDEGsfromthethreecomparisons:PLvsPN,PLvsC,andPNvsC(FC>1.
5or1.
5)and7downregulated(FC8).
STRTlibrariesofPLandPNsampleswerepreparedandsequencedatthesametimeascontrols12.
Thesampleswerepreparedintothreedifferentlibraries,asdemonstratedinSupplementaryTableS1.
TotalRNAsamples(threereplicatesforeachsample,10ngofeachreplicate)wereusedforRNAsequencinglibrarypreparationaccordingtotheSTRTprotocol10,whichwasadjustedfor10ngsamples.
ThelibrariesweresequencedusinganIlluminaHiSeq2000instrument.
RedundancywasreducedaccordingtoUMI46,andthenon-redundantreadsweredemultiplexedandtrimmedbydemlttoolinruby-bio-gadget(https://github.
com/shka/ruby-bio-gadget).
Thedemultiplexedreadswerealignedtohg19humanreferencegenome,ArrayControlRNAspikesandhumanribosomalDNAcompleterepeatingunit[GenBank:U13369]byTopHat47.
ThealignedSTRTreadswereassembledbysampletypesusingCufflinks48,and5′-endregionsoftheassembledtranscriptsweremergedasTranscriptFar5′-ends(TFEs).
TFEswerecomparedbyCufflinkswithUCSCgenestoannotate.
ReadsalignedwithintheTFEswerecountedbysamplesagain,andnormalizedbythespike-inbasednormalization11.
DifferentialexpressionanalysiswasperformedbySAMstrt11,12.
InRNAseqdata;extremelyhighFCvaluessuchas1*108resultsfromthecalculationmethodusedinSAMstrt.
Inthecomparisonbetweenzerotranscriptsandsometranscriptstoavoidcalculationerrorsbyzerodivision,SAMstrtaddssmallrandomnumberstoallnormalizedtranscriptcountsandthencalculatestheFC.
WeperformedPCAwiththescalingbutnon-centeringpreprocesssteps.
CorrelationofgeneexpressionwithPCwasestimatedbySAMstrtquantitativeresponsetest.
ScoresofsamplesonaPCweregivenasthequantitativevalues,andthresholdofthesignificantlycorrelatedgeneisLocal-FDR<1%.
QuantitativePCR.
cDNAsynthesiswascarriedoutusingrandomprimersandSuperScriptIIIFirst-StrandsynthesissystemforRT-PCR(18080-151,Invitrogen)accordingtomanufacturer'sinstructions.
10–20ngofcDNA(RNA)wasappliedtoeachqPCRassayandeachsamplewasruninthreereplicates.
qPCRwascarriedoutusinganABIPRISM7900HTSequenceDetectionSystemwithFastSYBRGreenMastermix(4385617,bothfromAppliedBiosystems)accordingtomanufacturer'sinstructions.
TheprimersequencesforPYCARD,CARD6,IFI16,andIL8areshowninSupplementaryFigureS3a.
RPL13andGAPDHwereusedasreferencegenesfornormalization.
Immunohistochemistry.
Formalinfixedparaffinsections(5um)werestainedwithdifferentanti-humanantibodiesusingtheperoxidase-basedImmPRESSReagentkit(VectorMP-7500)andImmPACTTMDABperoxidasesubstratekit(VectorSK-4105).
Epitoperetrievalwascarriedoutbyaheat-mediatedmethodin11sodiumcitratebufferpH6.
0for20minutes.
Theprimaryantibodieswere:ASC(AdipoGen,AG-25B-0006-C100),TMS1(Proteintech,10500-1-AP),CARD6(NovusBiologicals,NBP2-15704),IFI16(NovusBiologicals,NBP1-83118),IFI16(Abcam,ab55328),NLRP10(NovusBiologicals,NBP1-85556),andHumanin(ThermoFisher,PA1-41325).
NormalrabbitIgGwasusedasanegativecontrol(Dako,X0903).
Immunofluorescence.
Forimmunofluorescencestudies,weculturedcellsextractedfromfull-thicknesssamples12.
ThecellsweregrownoncoverslipswithRatTailCollagenI(Gibco,Invitrogen)coating,andfixedwithmethanolfor5minat20°C56.
ThecoverslipsampleswereincubatedonehouratroomtemperaturewiththeMTCO2(Abcam,ab3298)andCARD6antibodies.
AlexaFluor555and488conjugatedIgGs(Invitrogen,MolecularProbes)wereusedassecondaryantibodiesandthenucleistainedwithDAPI(Sigma-Aldrich).
ThepicturesweretakenwithZeissLSM5Duoconfocalmicroscope.
Immunoelectronmicroscopy.
Skinbiopsieswerefixedwith4%paraformaldehyde-PBSsolutionfor6to12hfollowedbyimmersionin2.
3Msucrose-PBSsolution.
Sampleswerefrozeninliquidnitrogen,andthincryosectionswerecutwithaLeicaUltracutUCTmicrotome.
Sectionswerefirstincubatedin0.
1%glycine-PBS,thenin1%BSA,labeledwithantibodiesagainstPYCARDorCARD6followedbyincubationwithprotein-Agoldconjugate.
LabelingwasdetectedwithaTecnaiG2Spirittransmissionelectronmicroscope(FEICompany)andQuemesaCCDcamera(MSISGmbH).
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AcknowledgementsWeareindebtedtolateProfessorUlpuSaarialho-Kerewithwhomthisstudywasconceived.
WethankAuliSaarinen,AlliTallqvist,andPiaPekkanenfortheirexcellentexperthelp.
IEMexperimentsweredonebyBiocenterOuluEMCoreFacilityco-fundedbytheUniversityofOuluandBiocenterFinland.
ConfocalimagingwasdoneatMolecularImagingUnit,BiomedicumHelsinki,UniversityofHelsinki.
ThisstudywassupportedbyAcademyofFinland(130360;1255560),SigridJuséliusFoundation,TEKES,HelsinkiUniversityCentralHospitalResearchFunds(J.
Vuola,S.
SuomelaTYH2009233),FolkhlsanResearchFoundation,SwedishResearchCouncil,theKarolinskaInstitutetDistinguishedProfessorAward,KarolinskaInstitutetinternalgrants(2013fobi38281and2013p4re39181),andBiomedicumHelsinkiFoundation.
ThecomputationresourceswereprovidedbySNICthroughUppsalaMultidisciplinaryCenterforAdvancedComputationalScience(UPPMAX)(b2010037).
AuthorContributionsAllauthors(M.
H.
T.
,S.
K.
,T.
S.
,H.
A.
S.
,J.
V.
,K.
N.
,R.
S.
,A.
J.
,S.
L.
,S.
S.
,E.
K.
,J.
K.
andO.
E.
)wereinvolvedinthestudydesign.
S.
S.
andJ.
V.
recruitedandclinicallycharacterizedthestudysubjects,andsampledtheskinspecimen.
H.
A.
S.
,K.
N.
andT.
S.
preparedthesamplesfortheexperiments.
A.
J.
performedthelibrarypreparationandsequencingusingthemethodologydesignedbyS.
L.
S.
K.
wasresponsibleforthebioinformaticspipelineoftheRNAseqdata;M.
H.
T.
furtheranalyzedthedata;O.
E.
andM.
H.
TwereresponsiblefortheimmunostainingexperimentsandR.
S.
fortheIEMstudies.
M.
H.
T.
,J.
K.
andO.
E.
draftedthemanuscript.
Alltheauthorscontributedcriticaldiscussionandapprovedthefinalversionofthemanuscript.
AdditionalInformationSupplementaryinformationaccompaniesthispaperathttp://www.
nature.
com/srepCompetingfinancialinterests:Theauthorsdeclarenocompetingfinancialinterests.
Howtocitethisarticle:Tervaniemi,M.
H.
etal.
NOD-likereceptorsignalingandinflammasome-relatedpathwaysarehighlightedinpsoriaticepidermis.
Sci.
Rep.
6,22745;doi:10.
1038/srep22745(2016).
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