Mechanisms in Acne Inversa Inflammatory Disease: Pathogenetic Deficiency of IL-22 Contributes ...

13 Pages·2010·2 MB·English

Deficiency of IL-22 Contributes to a Chronic
Inflammatory Disease: Pathogenetic
Mechanisms in Acne Inversa
Kerstin Wolk, Katarzyna Warszawska, Conny Hoeflich,
This information is current as
Ellen Witte, Sylke Schneider-Burrus, Katrin Witte, Stefanie
of November 18, 2018.
Kunz, Annette Buss, Hans Joachim Roewert, Markus
Krause, Ansgar Lukowsky, Hans-Dieter Volk, Wolfram
Sterry and Robert Sabat
J Immunol published online 8 December 2010
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Published December 8, 2010, doi:10.4049/jimmunol.0903907
The JournalofImmunology
Deficiency of IL-22 Contributes to a Chronic Inflammatory
Disease: Pathogenetic Mechanisms in Acne Inversa
Kerstin Wolk,*,† Katarzyna Warszawska,* Conny Hoeflich,‡ Ellen Witte,*
x
Sylke Schneider-Burrus, Katrin Witte,* Stefanie Kunz,* Annette Buss,*
Hans Joachim Roewert,x Markus Krause,x Ansgar Lukowsky,x Hans-Dieter Volk,‡
Wolfram Sterry,x and Robert Sabat*,†
OverexpressionoftheTcellcytokineIL-22islinkedtothedevelopmentofsomechronicdiseases,butlittleisknownaboutIL-22
deficiencyinhumans.Asdemonstratedinthisstudy,acneinversa(AI;alsodesignatedasHidradenitissuppurativa)lesionsshow
a relative deficiency of IL-22 and IL-20, but not of IL-17A, IL-26, IFN-g, IL-24, or IL-1b. Moreover, AI lesions had reduced
expression of membranous IL-22 and IL-20 receptors and increased expression of the natural IL-22 inhibitor, IL-22 binding
protein.AIisachronicinflammatoryskindiseasewithprevalenceupto4%ofthepopulationandinwhichcutaneousbacterial D
o
persistencerepresentsanimportantpathogeneticfactor.Accordingly,wealsofoundarelativedeficiencyofantimicrobialproteins w
n
(AMPs) in AI lesions and a positive correlation between lesional IL-22 and IL-20 versus AMP levels. IL-22, like its tissue lo
a
d
celldownstreammediatorIL-20,upregulatedAMPsinreconstitutedhumanepidermisandwascriticalforincreasedAMPlevels e
d
underinflammatoryconditions.TherelativeIL-22deficiencyinAIwasnotlinkedtolesionalTcellnumbersorTh22/Th1/Th17 fro
subsetmarkersand-inducingcytokines.However,IL-10washighlyexpressedinAIlesionsandcorrelatednegativelywithIL-22 m
h
expression. Moreover, IL-10 inhibited IL-22 but not IL-17 production in vitro. The IL-10 overexpression, in turn, was not ttp
associated with an elevated presence of regulatory T cells but with the enhanced presence of an IL-10–inducing cytokine. ://w
We conclude that IL-22 deficiency may contribute to the pathogenesis of certain chronic disorders as postulated in this paper w
forAI. TheJournalofImmunology,2011, 186:000–000. w
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A m
cne inversa(AI;alsoreferredtoasHidradenitissuppu- dateofpurulence.Leftuntreated,AIresultsinthedevelopmentof
u
rativa) is a chronic inflammatory skin disease with a painful,fistulatingsinusesandwidescars. no
prevalence rate of up to 4% (1–6). It affects the inter- TheexactetiologyofAI,however,isunknown.Smoking,obe- l.o
rg
triginousskinofperianal,inguinal,andaxillarysites(2,5).Initially, sity, a respective genetic background, and hormonal factors may b/
theocclusionofapocrineorfollicularductsleadstostasisaswell playaroleinitsdevelopmentand/orcourse(5).Theassumptionof y
g
as to accumulation and propagation of bacteria in the apocrine cutaneousbacterialpersistenceinaffectedskinasacrucialpatho- u
e
hairfollicleunitfollowedbydilatationandruptureoftheunit,in- genetic factor is in line with the fact that an antibiotic treatment st o
flammation,andtissuedestruction.Thepersistenceofstreptococci wassuccessful inAIpatients (7). n
N
and staphylococci in obstructed and ruptured hair follicles main- Antimicrobial proteins (AMPs) play an important role in the o
v
tainsandbooststheinflammationandleadstoasuperfluousexu- cutaneous defense against bacteria and are produced by kerati- e
m
nocytes(8,9).ExamplesforAMPsinhumansincludeb-defensin b
e
(BD)1,BD2,BD3,andtheS100proteinsS100A7(alsoknownas r 1
8
psoriasin), S100A8 (calgranulin A), and S100A9 (calgranulin B) , 2
*InterdisciplinaryGroupofMolecularImmunopathology,Dermatology/MedicalIm- (8,10,11).AMPsareconstitutivelyexpressedinhumanepithelia, 01
munology,UniversityHospitalCharite´,D-10117Berlin,Germany;†ResearchCenter although at low levels, and are upregulated by bacterial products 8
Immunosciences, University Hospital Charite´, D-10117 Berlin, Germany; ‡Institute and specific mediators (8, 12–17). The increase of AMP expres-
of Medical Immunology, University Hospital Charite´, D-10117 Berlin, Germany;
and xDepartment of Dermatology, University Hospital Charite´, D-10117 Berlin, sionmay becrucial for theprevention or limitation ofcutaneous
Germany infectionsinsituationsinwhichtheepidermalbarrierisdisrupted
ReceivedforpublicationDecember7,2009.AcceptedforpublicationNovember1, (e.g., in wounds and some skin diseases). In this regard, the dif-
2010. ference in the expression of AMPs between psoriasis (Ps) and
ThisworkwassupportedbytheGermanMinistryofEducationandResearchandby atopic dermatitis (AD)is interesting and has beenintensivelyin-
ResearchGrantSA1868/2-1(toR.S.)fromtheDeutscheForschungsgemeinschaft
vestigated: in lesional Ps, skin AMPs are strongly upregulated,
(GermanResearchFoundation).
and infections rarely occur (18, 19). In contrast, the AMP up-
AddresscorrespondenceandreprintrequeststoDr.RobertSabat,Interdisciplinary
GroupofMolecularImmunopathology,Dermatology/MedicalImmunology,Univer- regulationinlesionalskinfromADpatientsissignificantlylower,
sityHospitalCharite´,CampusCharite´Mitte,Charite´platz1,D-10117Berlin,Ger- predisposingthosepatientstofrequentskininfectionswithGram-
many.E-mailaddress:[email protected]
positivebacteria (streptococci and staphylococci) (20–23).
Theonlineversionofthisarticlecontainssupplementalmaterial.
Theaimofthisstudywastoshedlightonthemolecularmech-
Abbreviationsusedinthis article:AD,atopic dermatitis; AI,acneinversa;AMP, anismsbehindtheweaknessofantibacterialdefenseinAI,which
antimicrobialprotein;BD,b-defensin;HPRT,hypoxanthinephosphoribosyltransfer-
ase 1; IHC, immunohistochemistry; IL-22BP, IL-22 binding protein; PK, protein may form the basis of future therapy options. To this end, we
kinase;Ps,psoriasis;qPCR,quantitativereal-timeRT-PCR;rh,recombinanthuman; analyzedtheAMPexpressioninAI,compareditwiththatofPsand
RHE,reconstitutedhumanepidermis.
AD, and searched for mechanisms underlying the observed de-
Copyright(cid:1)2010byTheAmericanAssociationofImmunologists,Inc.0022-1767/10/$16.00 ficiency inAMP expression intheAIpatients.
www.jimmunol.org/cgi/doi/10.4049/jimmunol.0903907
2 PATHOGENETICMECHANISMSINACNEINVERSA
Materials and Methods samples were taken for quantitative real-time RT-PCR (qPCR) analysis.
Thesestudieswereapprovedbytheregionalauthoritiesforprovisionson
Patients
labor,health,andtechnicalsafety,Berlin,Germany.
FormRNAexpressionanalysesandimmunohistochemistry(IHC),biopsies
qPCR
obtainedfromsurgicallyexcisedskinareas(AI)or4–6-mmpunchbiopsies
(Ps,AD,controlskin)wereobtainedfromaffectedskinofadultpatients IsolationoftotalcellularRNA,reversetranscriptionofmRNA,andquan-
withAI(18–61y[mean6SD:36614y],58%male;diseaseseverity: titative gene expression analysis by TaqMan real-time PCR either using
moderatetoseverewithinvolvementofatleasttwobodyareas;samples theABIPrism7700SequenceDetectionSystemortheSteponeplus(both
derivedfrominguinal[27%],axillary[45%],perianal/sacral[27%]skin; from Applied Biosystems, Weiterstadt, Germany) of human and mouse
30% of patients had isotretinoin therapy), chronic plaque Ps (23–82 y
samples was done as described previously (14, 25). Oligonucleotide se-
[mean6SD:49615y],67%male;diseaseseverity:83%moderateto quencesusedforexpressionanalysisofthehumangenesBD1,BD2,BD3,
severe; skin samples derived from leg [38%], trunk [35%], upper arm IL-4,IL-10,IL-20,IL-22,IL-24,IL-26,IL-10R1,IL-22R1,IL-10R2,IL-
[4%],hand[12%],feet[4%],head[3%],inguinal[4%];atleastonepatient 20R1, IL-20R2, IL-22 binding protein (BP), and hypoxanthine phos-
had local steroid and vitamin D therapy), and chronic AD (19 to 54 y phoribosyltransferase 1 (HPRT) are listed in Supplemental Table I. All
[mean 6 SD: 27 6 11 y], 33% male; severe skin involvement; skin otherdetectionsystemswerepurchasedfromAppliedBiosystems.
samplesderivedfromleg[67%],trunk[17%],arm[16%];50%ofpatients
hadlocalsteroidtherapy)fromnonlesionalskinofadultpatientswithPs IHC
(pairedtolesionalskinsamplesofPspatients)andfromhealthyskinof
adultcontrolparticipants(22to67y[mean6SD:46614y],82%male; FrozenEpiDerm-201tissueswereprocessedasdescribedpreviously(24).
skinsamplesderivedfromleg[18%],trunk[64%],axilla[18%]).Allskin SectionswerestainedwithamouseAbtohumanS100A7(clone47C1068;
biopsies were approved by the clinical institutional review board of the Acris,Herford,Germany)orthecorrespondingisotypecontrolmouseAb
Charite´ UniversityHospital,Berlin,Germany. tohumanIgG1(cloneDAK-G01;DakoCytomation,Hamburg,Germany).
DetectionofprimaryAbbindingwasperformedusingtheLSAB+System-
Cell culture APkit(DakoCytomation).Skinbiopsieswerefixedin10%neutralbuff- Do
ered formalin, embedded in paraffin, routinely processed, sectioned at 5 w
UnderdevelopedEpiDerm-201reconstitutedhumanepidermis(RHE)tis- mm, and stained with an Ab to CD3 (clone T3-4B5; DakoCytomation). nlo
sues(MatTek,Ashland,MA)wereculturedasdescribedpreviously(24).In DetectionofprimaryAbbindingwasperformedusingtheLSAB2System- ad
the first experimental setting, culture medium was supplemented or not HRPkit(DakoCytomation).Allsectionswerefinallycounterstainedand ed
(mcol)n,trrhoIl)L-w1i7tAhr(e1c0omngb/imnaln),trhhuIFmNan-g(r(h1)0ILn-g2/2m(l)2,0onrgr/hmTlN),Fr-haIL(-220ng(/2m0l)n,ga/ cseocvteiorendsawsadsepsecrrfiobremdepdreavtiaou1s:l2y0(02m4)a.gCnoifiucnatitniognoufsisntaginaendoTcuclaerllscoiunnstkining from
cytokinemixcontainingrhIL-22(1ng/ml),rhIL-17A(1ng/ml),rhIFN-g net. h
(0.1ng/ml),rhIL-20(5ng/ml),andrhTNF-a(0.1ng/ml),withthesame ttp
morixwwithiththtehesaemxceepmtiioxncoofntoanineionfgthvaerTyincegllccoyntcoeknitnreastiolancskoinfgILat-2e2ac(h0.t1im,1e,, ELISA ://w
and 10 ng/ml) at each time. After 72 h, culture medium was harvested HumanIL-22andIL-17AlevelsweremeasuredusingrespectiveELISA w
w
foorrmERLNIASAanaanlydsibs.ioIpnstihees sweceorendtaekxepneriamndenstanlaspe-tftrionzge,nRHfoErweietrheerstiImHuC- kqiutasnftrifioemdRus&inDgSEyLsItSeAmsk.itHsufmroamn MS1B0L0Aa7ndanBdiomSoouursceeSIAntAernlaetvieolnsalw,erere- .jim
latedornotwithrhIL-22for72h,and500mg/mlLPSfromEscherichia spectively.HumanBD2levelswereassessedbythehumanBD2ELISA m
coli 0111:B4 (Sigma-Aldrich, Taufkirchen, Germany) was added on the DevelopmentKitfromPeproTech(Hamburg,Germany). un
o
RHEsurfaceforthefollowing24h. Statisticalanalysis l.o
Human PBMCs were isolated from healthy donors and cultured as rg
described previously(25,26). Stimulationin the presence orabsenceof Dataarepresentedasthemean6SEM.Forfurtheranalyses,SPSS14.0 b/
rchmIL2;-1O0r(th1o0cnlogn/me,l)Cwilaasgd,oSnuelzubsaincgh,eiGtheerrmimanmy)o,bailniztie-dCDan3ti-ACbD3(1Ambg(1/cmmg2;/ smoifctweawreere(SaPnSaSly)zwedasuusisnegd.thReesMulatsnnf–roWmhiptanteiyenUts/tceosntt(rtowlop-atratiilceidp)a.nCtsoarrned- y gu
NOortrhdoecnlsotnaed)t,pGluesrmaanntiy-)C,Da2c8ytAokbin(e1mmigx/ccmon2;sisRti&ngDoSfyrshtIeLm-1s,bWaniedsbrhaIdLe-n2- lRaetisounltsswonereinavniatrloy-zteredatbeadseRdHoEn,PthBeMSCp,eaarnmdacno’nsvceonrtrioelnaatliopnrimcoaerfyfickieernat-. est on
(eachat10ng/ml),orcontrolmedium.Nonstimulatedcellsservedasthe tinocyte cultures were tested using the Wilcoxon matched-pairs signed- N
control.After72h,culturesupernatantswereharvestedforELISA. ranktest(two-tailed). ov
NaiveCD4+TcellswereisolatedbyMACSbasedonnegativeselection e
m
using the Naive CD4+ T cell isolation kitII (Miltenyi Biotec, Bergisch- b
Glelaasdtb9a7c%h,.GCeerlmlsawnye)r.eOeibtthaeirneddirepcutrliytieasnaolfyzCeDd4o+rCstDim45uRlaAte+dcfeolrls5wderweitaht RAIesskuinltlsesionsshowa relativedeficiencyofAMPs er 18
anti-CD3andanti-CD28mAb(anti-CD3fromOrthocloneOKT3,Cilag; , 2
andanti-CD28fromR&DSystemscoatedonthebottomofculturevessels Bacterialpropagationinobstructedandrupturedhairfolliclesre- 01
[each at 1 mg/cm2] or Dynabeads Human T-Activator CD3/CD28 beads presentsacrucialpathogeneticeventinthedevelopmentofAIskin 8
[Invitrogen,Karlsruhe,Germany;1bead/Tcell])inthepresenceofeither
lesions.WehypothesizedthatanattenuatedincreaseofAMPsmay
10ng/mlrhIL-12and5mg/mlanti-IL-4mAb(Th1),20ng/mlrhIL-4,and
beresponsibleforthesurvivalofbacteriaintheselesions.Therefore,
5mg/mlanti–IFN-gmAb(Th2),10ng/mlrhIL-1b,10ng/mlrhIL-6,10
ng/mlrhIL-23,5ng/mlrhTGF-b1.2,5mg/mlanti–IL-4mAb,and5mg/ml wefirstanalyzedtheexpressionofBDs(BD1,BD2,andBD3)and
anti–IFN-g mAb (Th17), 10 ng/ml rhIL-6, 10 ng/ml rhTNF-a, 5 mg/ml S100proteins(S100A7,S100A8,andS100A9)inAIlesionsand,as
anti–IL-4mAb,5mg/mlanti–IFN-gmAb,5mg/mlanti–TGF-b1.2mAb, acomparison,inlesionalskinfromPsandADpatientsaswellasin
and2mM6-formylindolo[3,2-b]carbazole(EnzoLifeSciences,Lo¨rrach,
healthyskinfromcontrolparticipants.Inlinewithpreviousresults
Germany) (Th22), or 10 ng/ml rhIL-10, 10 ng/ml rhTGF-b1.2, 5 mg/ml
anti–IL-4mAb,5mg/mlanti–IFN-gmAb,5mg/mlanti–TGF-b1.2mAb, from others and ourselves (8, 14, 15, 17, 20–23), we found con-
and10nMall-trans-retinoidacid(Sigma-Aldrich)(regulatoryThcells). stitutiveAMPexpressioninhealthyskinandastrongupregulation
Primaryhumankeratinocytes(Lonza,Vervier,Belgium)wereculturedas of all AMPs except of BD1 in lesions from Ps and AD patients
described previously (27). To study IL-20 induction, keratinocytes were
(Fig. 1). The upregulation was very strong in Ps, whereas AD
stimulatedornot(control)withrhIL-22eitherat10ng/mlfor42h,10ng/
lesionsshowedalowerupregulationofsomeoftheseAMPs:BD2,
mlfor2,6,18,and42h(kineticstudy),orat1,3.2,10,and32ng/mlfor
42 h (study of dose dependency). All recombinant cytokines were pur- S100A7,S100A8,andS100A9(Fig.1).Importantly,inAIlesions,
chasedfromR&DSystems. AMPswerealsopresent,butlevelsofactuallyallanalyzedAMPs
were clearly lower than those found in Ps lesions (Fig. 1). Fur-
Mice
thermore,theBD1,BD3,andS100A7levelswereevenlowerinAI
To study cutaneous AMPand cytokine expression, 14-wk-old female thaninAD.Finally,theexpressionofBD1wasevendownregulated
BALB/cmicewerei.p.injectedwitheither1mgrecombinantmouseIL-22
compared withhealthyskin.Ourdata showthatAIlesionsshow
(10mg/ml;R&DSystems),100mgLPSfromE.coli0111:B4(1mg/ml;
Sigma-Aldrich)orarespectivevolume(100ml)ofPBS(control).Before arelativedeficiencyofAMPexpressioncomparedwithPsskin,and
and 1, 3, 6, 24, and 48 h postinjection, mice were sacrificed, and skin thisdeficiencyisactuallybroaderthanthatobservedinADpatients.
TheJournal ofImmunology 3
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FIGURE1. AIskinlesionsshowarelativedeficiencyinAMPexpression.Biopsiesofhealthyskinfromcontrolparticipants(control;n=8)andofskin ://w
lesionsfrompatientswithPs(n=14),AI(n=7),andAD(n=8)wereanalyzedforAMPmRNAexpressionbyqPCR.Dataaregivenasthemean6SEMrelative w
totheexpressionofthehouse-keepinggeneHPRT(leftpanels).ThepvaluesthatwerecalculatedbytheMann–WhitneyUtestareindicated(rightpanels). w
.jim
m
u
AIlesionsshowreducedupregulationofIL-22andIL-20 Ourfurtherstudiesfocusedonthereasonwhytheupregulation n
o
expression ofAMPsinAIwasnotaspronouncedasinPs,inwhichtheanti- l.o
microbialcompetenceofthedisturbedskinbarrierisincreasedtoa rg
Immune mediators regulate AMP expression in the skin (9). level that almost perfectly prevents cutaneous infection. Interest- b/
HavingdemonstratedarelativedeficiencyofallanalyzedAMPin y
ingly,thecorrelationanalysisofthedataobtainedfromthesepatients g
AI lesions, we then examined the levels of cytokines that might u
e
revealedapositiveassociationbetweentheexpressionofIL-22and s
reeitghuelratTecAeMllsP(IeLx-p2r2e,sIsFioNn-ga,nIdLt-h1a7tAa,reILp-r2o6d)u,ckeedraptirneofceyretensti(aIlLly-2b0y, IL-20andtheexpressionofallAMPsexceptBD1(TableI).Incon- t on
trast,forIL-17A,IL-26,IFN-g,IL-24,orIL-1bversusAMPlevels N
IL-24), ormonocytes/macrophages(IL-24,IL-1b). o
Thelesionsofallpatientgroupsshowedasignificantupregula- nosignificantcorrelationswerefoundwiththeexceptionofthose vem
betweenIL-17AandBD2andbetweenIL-17AandBD3(TableI).
tion of almost all analyzed cytokines compared with the healthy b
e
skin(Fig.2).However,whencomparingtheexpressionlevelsbe- r 1
IL-22andIL-20 induceAMPproductionbyreconstituted 8
wtweereensiAmIilaanrd(IPLs-,1t7hAos,eILo-f2I6L,-I1F7NA-,gI,LIL-2-62,4I)FoNrh-gig,hILer-2(I4L,-a1nbd)IiLn-A1bI, epidermis , 201
8
whereastheexpressionofIL-22andIL-20wassignificantlylower The data described above led us to hypothesize that the relative
inAI.Interestingly,theexpressionofIL-22andIL-20inAIlesions deficiencyofIL-22andIL-20isresponsiblefortherelativeAMP
wasevenlowerthaninAD(Fig.2). deficiency in AI lesions. We therefore analyzed the regulation of
IL-22andIL-20,althoughproducedbydifferentcelltypes(IL-22 AMP expression by these cytokines in vitro and in vivo. As
asaTandNKcellcytokine,andIL-20beingproducedbyactivated expected,treatmentofRHE,aninvitromodelmimickinghuman
keratinocytes and dendritic cells) are two structurally and func- skin(35),withIL-22orIL-20resultedinastrongupregulationof
tionallyrelatedcytokines(28,29).Thereceptorcomplexesofboth BD1, BD2, BD3, S100A7, S100A8, and S100A9 mRNA (Fig.
cytokinesareknowntobehighlyexpressedinkeratinocytesandare 4A), which was confirmed at the protein level for BD2 (Fig. 4B)
each composed of two different subunits: IL-22R1/IL-10R2 (re- and S100A7(Fig.4B,4D).
ceptorcomplexforIL-22),IL-20R1/IL-20R2(receptorcomplexI As representatives of those cytokines upregulated similarly in
forIL-20),andIL-22R1/IL-20R2(receptorcomplexIIforIL-20) AIandPslesions,wealsoincludedIFN-gandIL-17Aintheseana-
(28, 29). For IL-22, there is also a soluble, single-chain receptor lyses. IFN-g did not change BD2 or S100A7 production in RHE
calledIL-22BPthatactsasahigh-affinityinhibitorofIL-22activity (Fig. 4B–D), although it induced the chemokines CXCL9,
(30–34).Inthenextinvestigations,wefurtherfocusedonthedif- CXCL10, and CXCL11 (Fig. 4C and data not shown). IL-17A,
ferencesbetweenAIandPsandanalyzedthecutaneousexpression whichalsohasbeendescribedtoincreaseAMPexpression(16),in-
of these receptor subunitsinthese diseases. Interestingly,the ex- ducedBD2andS100A7productioninRHEcomparablytoIL-22
pression of IL-22R1, IL-20R1, and IL-20R2 was decreased (Fig. (Fig. 4B, 4D). This suggested that in vivo during skin inflam-
3A),whereasIL-22BPwasexpressedhigherinAIthaninPslesions mationdifferentcytokinesmayupregulateAMPsandthatanatten-
(Fig. 3B). This therefore should further reduce the possibility of uatedincreaseoftwomediators(IL-22andIL-20)doesnotneces-
IL-22andIL-20toactintheAIlesions. sarilyimplyalimitedAMPupregulation.Therefore,wenextapplied
4 PATHOGENETICMECHANISMSINACNEINVERSA
FIGURE 2. The upregulation of cutane-
ous IL-22 and IL-20 expression in patients
with AI is lower than that in Ps patients.
Biopsies of healthy skin from control par-
ticipants(control;n=8)andofskinlesions
frompatientswithPs(n=14),AI(n=7),
andAD(n=9)wereanalyzedforcytokine
mRNAexpressionbyqPCR.Dataaregiven
as the mean 6 SEM relative to the house-
D
keepinggeneHPRT(topandmiddlepanels). o
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The p values that were calculated by the n
Mann–WhitneyUtestareindicated(bottom lo
a
panel). de
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amixofcytokinesthatincludedIL-22,IL-17A,IFN-g,IL-20,and stimulation system may be considered as a model reflecting the g
u
TNF-a at concentrations that should mimic the situation in Ps difference between the Ps lesion (high IL-22 levels) and the AI e
s
lesions to the RHE cultures and observed a high upregulation of lesion(lowIL-22levels). t o
n
BD1,BD2,BD3,S100A7,S100A8,andS100A9aswellasofthe Next,weinvestigatedtheinfluenceofbacterialproductsonAMP N
chemokineCXCL2(Fig.4E,4F).WhenleavingoutindividualTcell expression. Surprisingly, an E. coli LPS isolate that additionally ov
e
cytokinesfrom the cytokine mix, IL-22 had the strongest overall containedproteinandnucleicacidcontaminationsdidnotinduce m
b
effectontheAMPexpression,whereasIL-17wasespeciallycriti- AMPexpressioninRHEbutupregulatedtheexpressionofIL-1b e
calforBD2expression(Fig.4E,4F).Intheseexperiments,acon- (Fig. 4H). r 1
8
centrationof1ng/mlIL-22wasapplied.Wethentestedtherelev- Interestingly,evenasinglei.p.applicationofmIL-22inBALB/c , 2
0
anceofdifferentIL-22concentrations(10,1,and0.1ng/ml)inthe mice strongly upregulated the cutaneous expression of mBD3 1
8
cytokine mix. As shown in Fig. 4G, decreasing IL-22 concen- (whichcorrespondstohumanBD2)andmS100A8(mS100A7has
trations led to decreasing BD2 production by these cultures. A not been identified so far) (Supplemental Fig. 1A). LPS applica-
high versus a low concentration of IL-22 in that multicytokine tion that induces high systemic (14) as well as high cutaneous
FIGURE3. AIpatientsshowreducedcutaneousexpressionofIL-22andIL-20receptorsubunits,butincreasedexpressionofIL-22BPcomparedwithPs
patients.BiopsiesofskinlesionsfrompatientswithPs(n=14)andAI(n=7)wereanalyzedforcell-associated(A)andsoluble(B)cytokinereceptor
mRNAexpressionbyqPCR.Dataaregivenasthemean6SEMrelativetothehouse-keepinggeneHPRT.*p#0.05;**p#0.01;***p#0.001,Mann–
WhitneyUtest.
TheJournal ofImmunology 5
TableI. CorrelationanalysisofcytokineandAMPexpression
BD1 BD2 BD3 S100A7 S100A8 S100A9
IL-22 0.41(0.064) 0.69(0.001) 0.75(0.000) 0.51(0.019) 0.48(0.028) 0.61(0.003)
IL-17A 0.14(0.533) 0.57(0.007) 0.50(0.021) 0.34(0.136) 0.24(0.286) 0.41(0.066)
IL-26 0.16(0.567) 0.28(0.232) 0.27(0.236) 0.21(0.363) 0.08(0.741) 0.21(0.357)
IFN-g 20.13(0.659) 0.10(0.674) 0.25(0.298) 20.02(0.938) 0.05(0.845) 0.01(0.964)
IL-20 0.61(0.003) 0.79(0.000) 0.74(0.000) 0.71(0.000) 0.64(0.002) 0.79(0.000)
IL-24 20.33(0.148) 0.13(0.563) 0.23(0.313) 20.20(0.385) 20.27(0.232) 20.15(0.518)
IL-1b 20.40(0.071) 0.03(0.905) 0.12(0.610) 20.42(0.058) 20.39(0.079) 20.28(0.214)
CytokineandAMPmRNAexpressiondatafromskinlesionsofAIandPspatients(n=21)wereanalyzedbyqPCRandcorrelatedby
meansofSpearman’srankcorrelationanalysis.Foreachfield,theSpearman’srankcorrelationcoefficientand,inparenthesis,thepvalues
aregiven.Significantcorrelations(p#0.05)areinboldface.
(SupplementalFig.1B)levelsofvariousproinflammatorymediators, chemokines CCL17, CCL27, CXCL9, and CXCL8 was not sig-
includingmIL-1bandmTNF-a,ledtoacomparable(mS100A8)or nificantly different between AI and Ps lesions. However, the ex-
evenmuchlower(mBD3)cutaneousAMPupregulationwhencom- pression of CCL20 (attracting not only CCR6-positive T cell
paredwithIL-22injection(SupplementalFig.1A). subsetsbutalsodendriticcellsandmacrophages)andCCL22was
decreased and that of CXCL2 (attracting neutrophilic granulo- D
Relative deficiencyinIL-22expressionisnotcausedby cytes) was clearly increased in AI, matching very well with the ow
reducedTcellinfiltration clinical pictureof AIaspurulent chronic inflammation. nlo
a
OurdatasuggestedacausalrelationshipbetweentherelativeIL-22/ IL-10inhibitsIL-22productionandisoverexpressedinAI ded
IL-20 deficiency and the relative AMP deficiency in AI lesions lesions fro
compared with Ps lesions. We then asked for the mechanism(s) BecauseanotherpotentialcauseoftherelativeIL-22deficiencyin m
h
behindtheIL-22andIL-20deficiency.BecauseIL-22isproduced AI lesions could be a deficiency of cytokines essential for the ttp
by T cells in Ps lesions (36–38), we first analyzed the extent of generation/activationofIL-22–producingcells,weinvestigated ://w
T cell infiltration. Fig. 5 shows macroscopic (Fig. 5A) and mi- theexpressionofIL-12p35,IL-12/IL-23p40,IL-23p19,IL-6,and w
croscopic(Fig.5B)picturesofAIandPslesions,withindication TNF-a. As demonstrated in Fig. 6A, however, there was no de- w
of T cells in the microscopic picture by IHC staining of CD3. ficiency in the expression of Th22 cell-inducing cytokines in AI. .jim
m
ComparedtouninvolvedskinfromPspatients,theTcellnumbers In fact, cutaneous TNF-a expression was virtually identical be-
u
n
were significantly increased in both AI and Ps lesions (Fig. 5B, tween AI and Ps patients, and IL-6 was even higher in AI. In- o
5C).However,AIandPslesionsdidnotdifferinthisrespect(Fig. terestingly, IL-12p35 was strongly upregulated in AI compared l.o
rg
5B, 5C).Among T cells, IL-22 was described to be produced by withPslesions,matchingthehighexpressionofT-bet(Fig.5E)in b/
the Th1 (25), Th17 (16, 39), and very recently by the Th22 (40, AI. y
g
41) cell subsets. To our knowledge, there is no data available A further reason of the relative IL-22 deficiency in AI lesions u
e
s
showing direct comparison of these Th cell subsets’ capacity to couldbethepresenceofamediatorabletoinhibitTcellcytokine t o
produce IL-22 in humans so far. Our comparison of the IL-22 production. We therefore searched for cytokines for which ex- n N
production capacity of human T cell subsets generated starting pressionlevelsinAIandPslesionsnegativelycorrelatedwithIL- o
v
fromblood-derivednaiveCD4-positiveTcells(seeMaterialsand 22expressionlevels.Of11cytokinesanalyzed(IL-1b,IL-4,IL-6, em
Methods) demonstrated high production by Th22 cells, moderate IL-10, IL-17A, IL-20, IL-23p19, IL-24, IL-26, TNF-a, and IFN- b
e
productionbyTh1cells,butonlyminorproductionbyTh17cells g),onlytheexpressionofIL-10,amajorimmunosuppressive/anti- r 1
8
(although these cells highly secreted IL-17A) (Fig. 5D). Among inflammatory mediator (46), showed a significant negative corre- , 2
others, the IL-22–producing T cell subsets differ by their ex- lation with IL-22 levels (Fig. 6B). In fact, IL-10 expression was 01
8
pression of chemokine receptor and transcription factor patterns: significantly elevated in AI lesions compared with both Ps and
Th22 cells express CCR4 (the receptor for CCL17 and CCL22), healthyskin(Fig.6C).Importantly,IL-10inhibitedtheproduction
CCR6 (the receptor for CCL20), and CCR10 (the receptor for ofIL-22bybothTCR-stimulatedandcytokine-stimulatedPBMCs
CCL27)andcontainhighlevelsofthearylhydrocarbonreceptor; in vitro (Fig. 6D). Of note, IL-10 did not decrease the IL-17A
Th1 cells are characterized by their expression of CXCR3 (the secretioninthisexperimentalsetting;itevenclearlyenhancedthe
receptorforCXCL9,CXCL10,CXCL11)andT-bet,butoftenalso TCR-mediated IL-17A production in the presence of costimula-
expressCCR6; Th17 cells, apart from CCR4 and CCR6, express tionvia CD28 (Fig. 6D). The selective inhibitory effect of IL-10
retinoic acid-related orphan receptor gt (29, 40–45). We inves- onIL-22,butnotonotherThcellcytokineswasalsosupportedby
tigated the cutaneous expression of selected molecules distin- theobservedlackofnegativecorrelationsbetweenlesionalIL-10
guishing these T cell subsets. None of the molecules was de- versus IL-17AorIL-26 levels (Fig. 6B).
creased inits expressioninAIlesionscomparedwithPslesions: The overexpression of IL-10 in AI lesions, however, may not
whereas CXCR3, CCR4, CCR10, retinoic acid-related orphan be responsible for the relative IL-20 deficiency in AI lesions. In
receptor gt, and aryl hydrocarbon receptor expression did not contrasttoIL-22,IL-20ismainlyproducedbykeratinocytes(25,
differbetweenthesediseases,theCCR6andtheT-betexpression 47,48),andIL-10doesnotregulatethekeratinocytebiologydue
in AI lesions was even enhanced compared with Ps lesions (Fig. to the lack of one of the IL-10 receptor subunits, IL-10R1 (Fig.
5E).ThesedatapointtomechanismsunderlyingtherelativeIL-22 6E).Accordingly,IL-10levelsdidnotcorrelatewithIL-20levels
deficiency other than the degree of lesional infiltration of Th22, intheinflamed skin(data notshown).
Th1, or Th17 cells. We also measured the expression of chemo- Weveryrecentlygeneratedseveralresultsthatdescribeindetail
kines attracting these T cell subsets in comparison with other howIL-22inducedtheIL-20productioninkeratinocytesandthe
chemokines. As demonstrated in Fig. 5F, the expression of the potentialrelevanceofthiscascadeforthePspathogenesis(27).We
6 PATHOGENETICMECHANISMSINACNEINVERSA
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FIGURE4. IL-22andIL-20areimportantregulatorsofkeratinocyteAMPexpression.A–D,RHEcultureswerestimulatedwith20ng/mlrhIL-22,20ng/
mlrhIL-20,10ng/mlrhIL-17A,10ng/mlrhIFN-g,and2ng/mlTNF-aasindicatedorwereleftuntreated(controls)for72h.A,mRNAexpressionwas
analyzedbyqPCR.Datafromfiveexperimentsaregivenasthemean6SEMrelativetoHPRTexpression.B,SecretedBD1andS100A7wasanalyzedby
ELISA.Datafromsixexperiments(dataoncontrol,IL-20,IL-22;mean6SEM)oroneexperiment(dataoncontrol,IL-17,IL-22andoncontrol,IFN-g,
IL-22)aregiven.C,mRNAexpressionwasanalyzedbyqPCR.Datafromthreeexperimentsaregivenasthemean6SEMrelativetoHPRTexpression.D,
Cell-associatedS100A7expressionwasanalyzedbyIHC.Representativepicturesfromthreeexperimentsaregiven.Originalmagnification3100.E,RHE
cultureswerestimulatedornot(control)for72hwithamixof1ng/mlrhIL-22,5ng/mlrhIL-20,1ng/mlrhIL-17A,0.1ng/mlrhIFN-g,and0.1ng/ml
rhTNF-aorwiththesamemixwiththeexceptionofoneoftheTcellcytokineslackingateachtime.mRNAexpressionwasanalyzedrelativetoHPRT
expressionbyqPCR,anddatafromthreeexperimentsaregivenasthemean6SEMasthepercentofthemix-stimulatedgroup.F,RHEcultureswere
treatedasinE.BD2levelsinculturesupernatantweremeasuredbyELISA.Datafromthreeexperimentsareshown.G,RHEcultureswerestimulatedor
TheJournal ofImmunology 7
demonstrate in this study that this IL-22 effect in vitro became absence or reduction of only one special cytokine may cause a
visibleafteralready2handatevenlowIL-22concentrations(Fig. strong attenuation of AMP induction. This led us to suggest that
7A), and itsstatistical significance couldbe demonstrated forthe thelimitedIL-22andIL-20upregulationisresponsibleforlimited
stimulation with 10 ng/ml IL-22 during 42 h (Fig. 7B). We AMP levels inAIcompared withPslesions.
therefore hypothesized that the relative IL-20 deficiency in AI ApartfromAI,ADalsorepresentsaninflammatoryskindisease
lesions may be caused by the relative IL-22 deficiency. This as- withfrequentbacterialpersistenceintheaffectedskin(20–23).We
sumptionwassubstantiatedbyastrongpositivecorrelationofIL- demonstrated in this study that, surprisingly, the extent of the
20expressionwithIL-22(Fig.7C),butnotwithIL-17AorIL-26 relative AMP deficiency in AD was much lower than in AI. In
expression (data notshown)inAIand Pslesions. contrast to AI, AD patients show IL-22 levels comparable to Ps
patients(Fig.2)(14).Inthesepatients,however,therelativeIL-17
ReasonsforIL-10overexpression inAIlesions
deficiency,probablydependentonlownumbersofskin-infiltrated
PotentialmechanismsofenhancedIL-10expressioninAIinclude Th17 cells, and the IL-4 overproduction, may be the key factors
the elevated presence of IL-10–secreting T cells (e.g., regulatory for the reduced AMP expression (Fig. 2) (21, 49). These known
Tcells)andtheelevatedexpressionofmediatorsknowntoinduce factsaboutADfurthersupporttheideathat,despiteredundancyof
monocyticcellIL-10production.NodifferenceinlesionalFoxp3 thedifferentmediatorsininducingAMPs,thefailureofonlyone
expression,amarkerofregulatoryTcells,wasfoundbetweenAI ortwoofthemmaycausecutaneousantimicrobialincompetence.
and Ps patients (Fig. 7D). However, the higher cutaneous ex- The induction of AMPs by IL-22 and IL-20 is in line with
pression of IL-1b in AI lesions (Fig. 2) in combination with the previous observations from others and ourselves (12–14, 16, 17,
positivecorrelation betweenIL-1bandIL-10mRNAlevels(Fig. 50, 51) regarding these mediators. We expanded upon these ob-
D
7E) (which was the only statistically significant, positive corre- servations by illuminating the high significance of IL-22 as an o
w
lation found for IL-10 when analyzing cutaneous cytokine ex- inducer of AMPs in comparison with other inflammatory media- n
pressions)maypointtoanimportantroleofIL-1bininducingIL- tors and bacterial components. Recently, results from animal loa
d
10expression inAI. models demonstrated a very important role of IL-22 in the mu- ed
cosal defense against extracellular bacteria in the gut and lungs fro
Discussion (52, 53) and even against systemic infection with Salmonella m
h
IncontrasttootherchronicinflammatoryskindiseasessuchasPs, enterica (54). Interestingly, the significance of IL-22 in the de- ttp
AIpatientsgreatlysufferfrombacterialpersistenceintheaffected fense against pathogens seems to be dependent on the kind of ://w
skin (2, 5, 7). In this study, we show for the first time, to our affected tissue. Furthermore, the Mizoguchi group (55) showed w
knowledge,thattheAMPexpressionwassignificantlylowerinAI very nicely the local positive role of IL-22 in a mouse colitis w
lesions than in Ps lesions and even than in lesions from AD model and postulated the involvement of diminished IL-22 pro- .jim
m
patients. Because cutaneous AMPs represent a major line of de- duction inthepathogenesisof ulcerativecolitis.
u
n
fenseagainstskininfections,wespeculatethatthisrelativeAMP As mentioned above, IL-22 is mainly produced by various Th o
deficiency may be responsible for bacterial propagation in AI cellsubsets(25).Regardingthehumansystem,ourdatapresented l.o
rg
lesions. inthispapersuggeststhatTh22andTh1cellsaremoreimportant b/
This raised the question of what the potential mechanisms un- IL-22 producers than Th17 cells. Although we excluded reduced y
g
derlyingtherelativeAMPdeficiencyinAIlesionsare.Wefound skin infiltration by Th22, Th1, and Th17 cells and reduced cuta- u
e
s
several hints suggesting that it is due to a relative deficiency of neous expression of mediators essential for T cell subset t o
IL-22andIL-20:first,thelevelsofIL-22andIL-20,butnotofIL- generation/activation as being responsible for the relative IL-22 n
N
17A,IL-26,IFN-g,IL-24,orIL-1b,althoughincreasedcompared deficiencyinAIlesions,wefoundanincreasedexpressionofIL- o
v
with healthy skin, were diminished in AI lesions compared with 10 in these patients. The IL-10 levels negatively correlated with em
Ps lesions. Second, AI compared with Ps lesions showed a re- IL-22 expression in the inflamed skin. Furthermore, IL-10 partly b
e
duced expression of the membrane-associated receptors for IL- inhibited IL-22 production in stimulated lymphocytes in vitro. r 1
8
22 and IL-20 and a higher expression of the IL-22–neutralizing Because, in turn, IL-22 induces IL-20 in keratinocytes, we spec- , 2
soluble receptor IL-22BP. Third, of all investigated mediators, ulatethatinAIskinlesions,diminishedIL-20productionisare- 01
8
only the lesional levels of IL-22 and IL-20 correlated with the sult ofreduced IL-22 production.
levelsofall analyzed AMPs.Forth, IL-22 andIL-20 stronglyin- Afurtherveryinterestingquestionisthatregardingthecauseof
ducedcutaneousAMPexpressionbothinvitro(RHE)andinvivo the overexpression of IL-10 in AI lesions. Different factors may
(mice; only IL-22 was tested). Fifth, the absence or reduced playarole,including:nucleotidepolymorphismsoftheIL-10gene
concentration of IL-22 during RHE stimulation with a combina- (56,57);accumulationofregulatoryTcells(Foxp3-positivecells),
tion of several cytokines had the broadest negative effect on the of Tr1 cells (IL-10–secreting Foxp3-negative cells), or of highly
inductionof AMPs. IL-10–producingAPCs(58–64);andtheenhancedpresenceofIL-
It seems that in general, upon inflammation, skin upregulates 10–inducing cytokines. Having investigated the latter possibility,
AMP expression compared with healthy skin to compensate the the positive correlation between lesional IL-10 and IL-1b levels
higherriskofinfectionarisingduetoimpairedskinintegrity.Itis foundinthecurrentstudymayindeedpointtoaroleofIL-1bin
verylikelythatthecooperationofseveralcytokines(includingIL- upregulatingIL-10.Nicotine,whichislikelytobepresentinmost
22, IL-17A, IL-20, IL-1b, TNF-a) is responsible for this AMP AI patients because of the high prevalence of smokers, may be
upregulation. However, our in vitro study demonstrated that the another reasonfor theincreased IL-10production. In fact, trans-
not(control)for72hwithamixof5ng/mlrhIL-20,1ng/mlrhIL-17A,0.1ng/mlrhIFN-g,0.1ng/mlrhTNF-a,andvaryingconcentrationsofrhIL-22as
indicated.BD2levelsinculturesupernatantweremeasuredbyELISA.Datafromtwoexperimentsareshown.H,RHEcultureswerestimulatedornotwith
20ng/mlrhIL-22for72handwithorwithout500ng/mlLPSforthefollowing24h.Unstimulatedculturesservedasthecontrol.mRNAexpressionwas
analyzedrelativetoHPRTexpressionbyqPCR.Datafromoneexperimentaregivenasthepercentofthecontrol.*p#0.05,Wilcoxonmatched-pairs
signed-ranktest.
8 PATHOGENETICMECHANISMSINACNEINVERSA
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FIGURE5. ThecutaneousIL-22deficiencyinAIcannotbeattributedtoadifferentiallesionalTcellinfiltration.A,Macroscopicappearanceoflesions
fromaPs(toppanel)andanAI(bottompanel)patient.B,BiopsiesfromskinlesionsofPs(lefttoppanel)andAI(leftbottompanel)patientsandfrom
healthyskinfromcontrolparticipants(rightpanels)wereanalyzedforCD3-positivecellsbyIHC.Scalebars,200mm.C,EpidermalanddermalCD3-
positivecellnumbersinbiopsiesfromskinlesionsofAI(n=10)andPs(n=13)patientsandfromuninvolvedskinfromPspatients(controlskin;n=5)
wereanalyzedbyIHC.Dataaregivenasthemean6SEM.D,ActivatedThcellsubsetsweregeneratedfromnaivehumanTcellsandanalyzedforIL-22
andIL-17AproductionbyELISAofcellculturesupernatant.Datafromfiveexperimentsareshown(mean6SEM;*p#0.05comparedwithallother
Tcellsubsets,Wilcoxonmatched-pairssigned-ranktest).BiopsiesofskinlesionsfrompatientswithPs(n=12)andAI(n=7)wereanalyzedforthe
expressionofchemokinereceptorsandtranscriptionfactorsmarkingTcellsubsets(E)andofchemokines(F)byqPCR.Dataaregivenasthemean6SEM
relativetoHPRTexpression.*p#0.05;**p#0.01;***p#0.001,Mann–WhitneyUtest.
TheJournal ofImmunology 9
FIGURE 6. The regulation of lymphocyte IL-22
production by IL-10 may represent a pathogenetic
cascade in AI. A, Biopsies of lesional skin from
patients with Ps (n = 13 or 14) and AI (n = 7) were
analyzedforIL-12p35,IL-12/IL-23p40,IL-23p19,IL-
6, and TNF-a mRNA expression by qPCR. Data are
givenasthemean6SEMrelativetoHPRTexpression.
B, IL-10 mRNA expression data (obtained by qPCR D
relative to HPRT) were correlated with mRNA ex- ow
pressiondataofIL-22,IL-17A,andIL-26(obtainedby nlo
qPCRrelativetoHPRT)fromPsandAIlesions(n= a
d
21)bymeansofSpearman’srankcorrelationanalysis. ed
The Spearman’s rank correlation coefficients (rs) are fro
indicated.C,BiopsiesofcontrolskinfromPspatients m
(control;n=8),lesionalskinfromPspatients(n=14), h
and lesional skin from with AI patients (n = 7) were ttp
analyzedforIL-10mRNAexpressionbyqPCR.Data ://w
are given as the mean 6 SEM relative to HPRTex- w
w
ipmremssoiobnil.izeDd,aHntui-mCaDn3PABbMs,Casntiw-CerDe3sptilmusulaantetid-CwDi2t8h .jim
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Abs, and a cytokine mix consisting of rhIL-1b and u
n
rhIL-2 (each at 10 ng/ml) or were left unstimulated, o
eachinthepresenceandabsenceof10ng/mlrhIL-10, l.o
for 72 h. The concentration of IL-22 and IL-17A brg/
proteininculturesupernatantwasmeasuredbyELISA. y
Data from five experiments are givenas the mean 6 gu
SEM.E,RHEcultureswerestimulatedornot(control) es
for72hwithamixof1ng/mlrhIL-22,5ng/mlrhIL- t o
n
20,1ng/mlrhIL-17A,0.1ng/mlrhIFN-g,and0.1ng/ N
ml rhTNF-a. IL-10R subunit mRNA expression was ov
e
analyzed by qPCR. Data from five experiments are m
givenrelativetoHPRTexpressionasthemean6SEM. be
*p # 0.05, Wilcoxon matched-pairs signed-ranktest; r 1
8
**p#0.01;***p#0.001,Mann–WhitneyUtest. , 2
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1
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cutaneous nicotine administration in humans increased IL-10 causestrongactivationofthePKC/NF-kBpathway,nicotinemay
levelsafter LPS application (65). Very probably, monocyticcells increase cyclic 39,59-adenosin-monophosphate levels that induce
(66), the a4 and b2 subunits of nicotinic acetylcholine receptor activation of PKA. The simultaneous presence of IL-1b/TNF-a
(67),andtheincreaseofintracellularcyclic39,59-adenosin-mono- and nicotine may therefore mediate strong IL-10 production in
phosphate levels (68) are involved in this nicotine effect. Impor- AIlesions.
tantly, for a significant IL-10 secretion, the activation of protein In summary, we hypothesize an immunologically based path-
kinase(PK)CandNF-kBaswellasofthePKApathwayisnece- ogenetic cascade underlying the high frequency of bacterial
ssary (69). Whereas inAI lesions, theelevated IL-1blevels may infectionsinAI,whichincludesthefollowingsteps:1)increased