IL-17/Th17 Pathway Is Activated in Acne Lesions

18 Pages·2014·10.83 MB·English

IL-17/Th17 Pathway Is Activated in Acne Lesions
Hanna-Leena Kelha¨la¨1, Riitta Palatsi1, Nanna Fyhrquist2, Sari Lehtima¨ki2, Juha P. Va¨yrynen3,
Matti Kallioinen3, Minna E. Kubin1, Dario Greco2, Kaisa Tasanen1, Harri Alenius2, Beatrice Bertino4,
Isabelle Carlavan4, Bruno Mehul4, Sophie De´ret4, Pascale Reiniche4, Philippe Martel5, Carine Marty5,
Ulrike Blume-Peytavi6, Johannes J. Voegel4*, Antti Lauerma7*
1DepartmentofDermatology,UniversityofOuluandMedicalResearchCenter,OuluUniversityHospital,Oulu,Finland,2UnitofSystemsToxicology,FinnishInstituteof
OccupationalHealth,Helsinki,Finland,3DepartmentofPathology,UniversityofOuluandOuluUniversityHospital,Oulu,Finland,4Research,GaldermaR&D,Sophia
Antipolis,France,5EarlyDevelopment,GaldermaR&D,SophiaAntipolis,France,6DepartmentofDermatologyandAllergy,ChariteUniversita¨tsmedizin,Berlin,Germany,
7DepartmentofDermatology,UniversityofHelsinkiandHelsinkiUniversityCentralHospital,Helsinki,Finland
Abstract
Themechanismsofinflammationinacnearecurrentlysubjectofintenseinvestigation.Thisstudyfocusedontheactivation
of adaptive and innate immunity in clinically early visible inflamed acne lesions and was performed in two independent
patient populations. Biopsies were collected from lesional and non-lesional skin of acne patients. Using Affymetrix
Genechips, we observed significant elevation of the signature cytokines of the Th17 lineage in acne lesions compared to
non-lesionalskin.TheincreasedexpressionofIL-17wasconfirmedattheRNAandalsoproteinlevelwithreal-timePCR(RT-
PCR) and Luminex technology. Cytokines involved in Th17 lineage differentiation (IL-1b, IL-6, TGF-b, IL23p19) were
remarkablyinducedattheRNAlevel.Inaddition,proinflammatorycytokinesandchemokines(TNF-a,IL-8,CSF2andCCL20),
Th1 markers (IL12p40,CXCR3, T-bet, IFN-c),T regulatory cellmarkers (Foxp3, IL-10, TGF-b) and IL-17 related antimicrobial
peptides (S100A7, S100A9, lipocalin, hBD2, hBD3, hCAP18) were induced. Importantly, immunohistochemistry revealed
significantlyincreasednumbersofIL-17ApositiveTcellsandCD83dendriticcellsintheacnelesions.Insummaryourresults
demonstratethepresenceofIL-17ApositiveTcellsandtheactivationofTh17-relatedcytokinesinacnelesions,indicating
that the Th17 pathway is activated and may play a pivotal role in the disease process, possibly offering new targets of
therapy.
Citation:Kelha¨la¨H-L,PalatsiR,FyhrquistN,Lehtima¨kiS,Va¨yrynenJP,etal.(2014)IL-17/Th17PathwayIsActivatedinAcneLesions.PLoSONE9(8):e105238.
doi:10.1371/journal.pone.0105238
Editor:PierreBobe´,INSERM-Universite´Paris-Sud,France
ReceivedApril25,2014;AcceptedJuly17,2014;PublishedAugust25,2014
Copyright: (cid:2)2014 Kelha¨la¨ et al.This isan open-access article distributedunder thetermsof theCreativeCommonsAttribution License, which permits
unrestricteduse,distribution,andreproductioninanymedium,providedtheoriginalauthorandsourcearecredited.
DataAvailability:Theauthorsconfirmthatalldataunderlyingthefindingsarefullyavailablewithoutrestriction.DataareavailablefromtheNCBIGEO,withthe
accessionnumberGSE5379.
Funding:FundedbyOuluUniversityHospitalMedicalResearchCenter,byFinnishSocietyofDermatology,byHelsinki-UusimaaHospitalDistrictResearchFunds
andbyGaldermaR&Dinternalfunding.ThefunderGaldermaR&Dprovidedsupportintheformofsalariesforauthors(BB,IC,BM,SD,PR,PM,CM,JJV),butdid
nothaveanyadditionalroleinthestudydesign,datacollection,andanalysis,decisiontopublish,orpreparationofthemanuscript.Thespecificrolesofthese
authorsarearticulatedinthe‘authorcontributions’section.
CompetingInterests:ResearchersworkinginSophiaAntipolis,France,areGalderms’semployees.Thisdoesnotaltertheauthors’adherencetoPLOSONE
policiesonsharingdataandmaterials.
*Email:[email protected](AL);[email protected](JJV)
Introduction importance of TLR-mediated immune response is supported by
the presence of TLR2 expressing cells in inflammatory acne
Acne is a common disease characterized by androgen depen-
lesions. Furthermore, non-immune cells like keratinocytes and
dence, follicular hyperkeratosis, increased sebum excretion,
sebocytesexpressfunctionalTLR2[6–12].P.acnesisconsidered
colonization withPropionibacterium acnes(P.acnes) andinflam- tobeatriggerofexaggeratedTLR2mediatedimmuneresponses
mation. The earliest subclinical acne lesion is the microcomedo,
in acne [13]. TLR2 receptors are involved in the recognition of
which results from increased proliferation and retention of
wide array of microbial molecules mainly in gram-positive
infundibular keratinocytes [1]. The cytokine IL-1a may have a
bacteria, and also yeasts [14]. Recently, P. acnes was shown to
role in the initiation of microcomedos, by its ability to induce
activateNod-likereceptor3(NLRP3)inflammasomeinmonocytic
hypercornification of keratinocytes [2,3]. The formation of
cellsleadingtotheproductionofIL-1b[15,16].However,itisstill
microcomedos is preceded by mononuclear infiltrates consisting
unclear whether P. acnes can initiate comedogenesis or early
mainly of CD4+ T-cells and CD68+ macrophages [3]. CD4+ T-
phaseinflammatoryreactioninacne[17].Alsoothertriggersthan
cells are the major leukocytes in the early (6–72h) inflammatory
P. acnes for the early inflammatory cascades in acne lesion
infiltrates inacnelesions,withasmallportionofCD1+dendritic
formation for example leukotriens or free fatty acids should be
cells. Neutrophils emerge increasingly numerous in the 24h and
considered[18].
72hlesions,whicharethenclinicallyclassifiedaspustules.Atlater
In addition to innate immunity, also adaptive immunity, and
time pointsCD8+cells infiltrate inthelesions [4,5].
especially the Th17 pathway, may contribute significantly to the
IthasbeensuggestedthatP.acnesisinvolvedinthetriggering
inflammatoryresponseinacne[18],[19].PreviouslyP.acneshas
of inflammatory acne via Toll-like receptors (TLRs). The
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Th17PathwayinAcne
Table1. Clinicalcharacteristics ofacnepatient groupfrom Finland.
Finnishpatients(n=20)
Subject Age1 Gender Acneseverity2 Technology3
1 21 female 2 RT-PCRandimmunohistochemistry
2 24 female 1
3 21 female 2
4 17 female 3
5 34 female 1
6 25 female 1
7 29 female 2
8 22 male 3
9 29 male 3
10 21 male 2
11 26 female 2
12 22 male 3
13 19 male 2
14 27 male 3
15 25 male 1
16 26 male 2
17 25 male 4
18 36 female 1
19 31 female 2
20 18 female 2
Mean 24.9±5.1
1Dataareexpressedasmean6SD.
2Acneseverityatthebiopsysite(backorchest)wasassessedbyLeedsrevisedacnegradingsysteminFinnishpatientgroup.
3Thetechnology,whichwasusedtoinvestigatethebiopsiestakenfromthepatients.
doi:10.1371/journal.pone.0105238.t001
been shown to stimulate the production of IL-17A and IFN-c in Subjects and sampling
peripheralbloodmononuclearcells(PBMCs)[20–22].Moreover, The study was performed in two clinical centers in Oulu,
the increased expression of cytokines and other inflammatory Finland and Berlin, Germany. A total of 56 acne patients with
markerssuchasIL-1a,beta-defensins1and2,TNF-a,IL-1b,IL- moderate to severe acne vulgaris were included in the study.
8, IL-10, matrix metalloproteinases MMP-1, MMP-3, MMP-9, Clinical characteristic of patients is presented in Tables 1 and 2.
CXCL-2 was foundinacne lesions invivo[3,23–26]. Control subjects comprised patients with psoriasis (n=9, age
This study was based on the analyses of skin biopsies from range28–65;mean52.9,notage-matched)andhealthyvolunteers
clinically early looking inflamed acne lesions (comedones with thathadneverhadacne(n=6,agerange23–36;mean26.5).The
minimalerythematosusflareorsmallpapules).Thestudymaterial inclusion criteria for psoriasis patients were chronic plaque
was recruited by two clinical centers - Oulu, Finland and Berlin,
psoriasis, not receiving systemic or UV-treatment for at least 1
Germany-withindependentgroupsofpatientswithacnevulgaris,
monthbeforesampling.3mm(Germancohort)or4 mm(Finnish
aswellaspsoriasispatientsandhealthyvolunteersascontrols.Our
cohort)punchbiopsieswereobtainedunderlocalanesthesiafrom
resultsshowthat,asinpsoriasis,theTh17pathwayissignificantly
the back or chest of acne patients from lesional and non-lesional
up-regulatedbothattheRNAandproteinlevelinlesionsofacne
skin.Theacnelesionsobtainedbybiopsywereidentifiedclinically
vulgaris. The results suggest a novel pathomechanism in inflam-
as early inflammatory acne lesions meaning comedones which
matory acne, and open up the possibility for a new class of
were shading into small red papules (Figure1). The control skin
therapeutics targeting theTh17system insevere acne.
biopsiesweretakenfromthebackofpsoriasispatientslesionaland
non-lesional skin and healthy volunteers back skin. Utilization of
Materials and Methods
all biopsiesisdescribedin Figure S1.
Ethics statement
Affymetrix
Thestudiespresentedinthismanuscripthavebeenapprovedby
Biopsies for the microarray analysis were stored in RNA
local Ethics committees of Oulu University Hospital in Finland
and the Charite Universita¨tsmedizin Berlin in Germany. The Stabilization Reagent. For RNA extraction the samples were
biopsiesweretakenwithinformedandwrittenconsent.Allclinical homogenizedwithapotterinQiagenlysisbuffer.TotalRNAwas
investigationswereconductedinaccordancewiththeDeclaration extracted using miRNeasy extraction kits according to manufac-
of Helsinki Principles. turer’s protocol. RNA quantity was measured using Nanodrop
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Table2. Clinicalcharacteristics ofacnepatient groupfrom Germany.
Germanpatients(n=36)
Subject Age1 Gender Acneseverity2 Technology3
1 18 male Severe AffymetrixandRT-PCR
2 19 male Severe
3 20 female Moderate
4 21 male Severe
5 28 male Moderate
6 26 male Severe
7 22 male Severe
8 30 male Mild
9 23 male Severe
10 24 male Moderate
11 19 male Severe
12 26 male Mild
13 25 male Moderate Luminex
14 20 male Severe
15 28 male Moderate
16 27 male Moderate
17 21 male Moderate
18 22 male Mild
19 25 male Moderate
20 19 male Severe
21 20 male Mild
22 29 female Moderate
23 31 male Severe
24 31 male Moderate
25 18 male Severe Immunohistochemistry
26 19 male Moderate
27 29 male Severe
28 24 male Severe
29 30 male Severe
30 20 male Moderate
31 19 male Moderate
32 19 female Moderate
33 27 female Severe
34 29 male Moderate
35 22 male Severe
36 21 male Moderate
Mean 23.6±4.2
1Dataareexpressedasmean6SD.
2Acneseverityatthebiopsysite(back)wasassessedbyGlobalEvaluationAcnescaleinGermanpatientgroup.
3Thetechnology,whichwasusedtoinvestigatethebiopsiestakenfromthepatients.
doi:10.1371/journal.pone.0105238.t002
spectrophotometerND8000.RNAqualitywasmonitoredusinga (OmicSoft, Cary, NC). Mean expression levels were obtained by
2100 Bioanalyzer (Agilent Technologies, Waldbronn, Germany). calculating the geometrical means of the RMA-normalized data
ProbesweresynthesisedandthenhybridizedonAffymetrixU133 forinvolvedandnon-involvedsamplegroups,respectively.Atwo
Plus 2.0 chips (Affymetrix, Santa Clara, CA). All chips were sided paired T test was performed using Array Studio (OmicSoft
normalized using RMA method [27]. Only Affymetrix identifiers Corporation, USA), to determine which genes were significantly
(IDs) with expression $2exp6 in at least one condition were differentially expressed between involved and non-involved
selected. Finally, 38014 out of 54675 initially present IDs were groups, and Benjamini-Hochberg false discovery rate (FDR)
selected. Data analysis was performed on Array Studio software
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Figure1.Macroscopicappearanceofbiopsiedlesionsfromacnepatients.
doi:10.1371/journal.pone.0105238.g001
multiple testing correction [28] was applied. The raw data is Kit)(Bio-Rad Laboratories, Hercules CA). Cytokines were quan-
available at NCBIGEO,accessionnumber GSE53795. tified in duplicate using following Luminex assays (Life Technol-
ogies, Carlsbad, CA): Human Cytokine, Premixed 23 Plex,
RT-PCR analysis Immunoassay Procarta kit and Milliplex Human Cytokine,
SnapfrozenskinbiopsieswerehomogenizedinTrisure(Bioline, Premixed 42 Plex, Immunoassay kit (Merck Millipore, Billerica,
London, UK) and RNA was extracted according to standard MA). Cytokine quantitities were normalized to the total concen-
protocols and subjected for cDNA synthesis. Complementary trationof protein.
DNA synthesis, and RT-PCR analysis were performed as
previouslydescribed [29]with7500FastRealTime PCR-system Histology and immunohistochemistry
(Applied Biosystems, Foster City, CA) in Finland and Applied Biopsies for histological analysis were fixed in 10% buffered
Biosystems 7900HT machine in France. The data were analysed formaldehyde. After dehydration/impregnation and inclusion in
by using the 22(DDCt) method, according to the manufacturer’s paraffin, 4 mm-thick serial sections were prepared. Slides were
instructions.StatisticalanalyzesweredonewithStudent’st-testor stained usingHematoxylin-Eosin.
when variances were significantly different, with nonparametric For immunohistochemistry of German patient samples, depar-
Mann-WhitneyUtestusingGraphPadPrismsoftware(GraphPad affinised sections were treated for antigen retrieval in 10mM
Software Inc.,LaJolla, CA). citrate buffer, pH 6.0 at 98uC for 20 minutes. Mouse antihuman
CD3 (clone Ab-9, Thermo Scientific, Fremond, CA) was used to
Cytokine profiling by Luminex technology detect T-lymphocytes. IL-17A was detected on the same section
Cytokines were extracted from non-lesional and lesional skin withagoatpolyclonalantihumanIL-17Aantibody(R&Dsystems,
which were crushed manually for 4 minutes with a glass potter Minneapolis, MN). CD3 was detected with Alexa fluor 594
(Dominique Dutscher S.A, Brumath, France) in 200mL ice-cold conjugatedantibodyandIL-17Awasrevealedafteramplification
PBS containing Triton X100 0, 2% and protease inhibitors (Biotin/ Streptavidin FITC). Positive cells were counted visually
(cOmplete, Mini, EDTA-free) (Roche Applied Science, Mann- on slides scanned withtheNanoZoomer (Hamamatsu, Japan).
heim, Germany). Samples were stirred for 30 minutes at 4 u, at For immunohistochemistry of Finnish samples, heat-induced
1400RPMandthencentrifugedfor5minutesat4uC,at 10000 antigenretrievalofthedeparaffinisedsectionswasperformedina
RPM. Supernatants were harvested and the concentration of microwaveoven.Endogenousperoxidaseactivitywasneutralized.
proteins was determined by colorimetric method (Bio-Rad DC Thesectionswereincubatedwithmonoclonalmouseanti-human
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Table3. Top20up-regulated genesin the Germanacne patientgroup based onAffymetrix microarraytechnology.
Foldchange
Meanexpression (involvedvs.
ProbeSet Genesymbol Title (non-involvedskin) non-involvedskin) FDR
232170_at S100A7A S100calciumbindingproteinA7A 235 29 ,0.001
202859_x_at IL8 interleukin8 162 25 ,0.001
207356_at DEFB4 defensin,beta4 451 25 ,0.001
211906_s_at SERPINB4 serpinpeptidaseinhibitor,cladeB 768 19 ,0.001
(ovalbumin),member4
204475_at MMP1 matrixmetallopeptidase1(interstitial 124 19 ,0.001
collagenase)
212768_s_at OLFM4 olfactomedin4 65 16 0.0018
205828_at MMP3 matrixmetallopeptidase3(stromelysin1, 79 16 ,0.001
progelatinase)
204470_at CXCL1 chemokine(C-X-Cmotif)ligand1 51 15 ,0.001
(melanomagrowthstimulatingactivity,alpha)
210873_x_at APOBEC3A apolipoproteinBmRNAeditingenzyme, 53 14 ,0.001
catalyticpolypeptide-like3A
205863_at S100A12 S100calciumbindingproteinA12 47 14 ,0.001
203691_at PI3 peptidaseinhibitor3,skin-derived 987 13 ,0.001
209875_s_at SPP1 secretedphosphoprotein1 96 13 ,0.001
206561_s_at AKR1B10 aldo-ketoreductasefamily1,memberB10 135 13 ,0.001
(aldosereductase)
205681_at BCL2A1 BCL2-relatedproteinA1 81 13 ,0.001
206336_at CXCL6 chemokine(C-X-Cmotif)ligand6(granulocyte 13 11 ,0.001
chemotacticprotein2)
209774_x_at CXCL2 chemokine(C-X-Cmotif)ligand2 54 11 ,0.001
204580_at MMP12 matrixmetallopeptidase12(macrophage 67 11 ,0.001
elastase)
205114_s_at CCL3/CCL3L1/ chemokine(C-Cmotif)ligand3/chemokine 65 10 ,0.001
CCL3L3 (C-Cmotif)ligand3-like1/chemokine
(C-Cmotif)ligand3-like3
210146_x_at LILRB2 leukocyteimmunoglobulin-likereceptor, 32 10 ,0.001
subfamilyB(withTMandITIMdomains),
member2
210413_x_at SERPINB3/SERPINB4 serpinpeptidaseinhibitor,cladeB 1936 10 ,0.001
(ovalbumin),member3/serpinpeptidase
inhibitor,cladeB(ovalbumin),member4
doi:10.1371/journal.pone.0105238.t003
antibodiestoCD4(clone4B12,1:50,Novocastra,Newcastle,UK), (0.14mm2). Data are presented as median positive cell count
CD8 (clone 4B11, 1:200, Novocastra), CD68 (clone KP-1, (interquartile range). The statistical analysis was performed by
1:10000, Dako, Copenhagen, Denmark), CD83 (clone 1H4b, IBMSPSSStatistics19(IBM,Chicago,IL).Mann-WhitneyUtest
1:25, Dako), Foxp3 (clone 236A/E7, 1:100, Abcam Ltd., Cam- two-tailed p-value less than 0.05 was considered statistically
bridge, UK), T-bet (clone 4B10, 1:200, Abcam Ltd.) and goat significant.
polyclonal anti-human antibody to IL-17A (1:100, R&D systems,
Minneapolis, MN). Bound antibodies were detected using the Results
NovoLinkPolymerdetectionsystem(LeicaBiosystems,Newcastle,
UK), the EnVision system (Dako) or Goat-HPR-Polymer kit Genome-wide transcriptome reveals Th17-related
(Biocare Medical, Concord, CA). Deaminobenzidine (DAB) was signaling significantly elevated in acne lesions
used as the chromogen and hematoxylin as the counterstain. Thestudywasperformedintwoseparatepatientcohortsfrom
Digital image analysis was used to count the positive cells. The Germany and Finland (Tables 1 and 2). The gene expression in
digital images were obtained under 206magnification from all skin biopsies of the subjects recruited in Germany was screened
sections with the use of a Nikon Eclipse E600 microscope and a using Affymetrix microarray technology. In the array analyses, a
Olympus DP25 digital camera. Images were taken from upper total of 904 genes were differentially expressed in acne lesions
dermalareaandaroundpilosebaceousfollicles,resultingin2to7 compared with non-lesional skin, and 509 of these were
images per slide. Image analysis was performed as described upregulated, whereas 395 were downregulated (Tables 3 and 4).
previously using ImageJ software [30] to record the average All microarray data have been deposited in NCBI’s Gene
number of the positively stained cells per 206 field image ExpressionOmnibus[31]andareaccessiblethroughGEOseries
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Table4. Top20down-regulated genes inthe German acne patientgroup basedon Affymetrixmicroarray technology.
Mean Foldchange
expression (involvedvs.
(non-involved non-involved
ProbeSet Genesymbol Title skin) skin) FDR
204515_at HSD3B1 hydroxy-delta-5-steroiddehydrogenase,3beta-andsteroid 2897 28.8 0.002
delta-isomerase1
205380_at PDZK1 PDZdomaincontaining1 1914 26.4 0.002
210576_at CYP4F8 cytochromeP450,family4,subfamilyF,polypeptide8 1305 26.3 0.002
241412_at BTC betacellulin 791 26.1 ,0.001
234513_at ELOVL3 elongationofverylongchainfattyacids(FEN1/Elo2,SUR4/Elo3, 8094 26.1 0.003
yeast)-like3
1553583_a_at THRSP thyroidhormoneresponsive(SPOT14homolog,rat) 5008 25.9 0.003
206143_at SLC26A3 solutecarrierfamily26,member3 166 25.7 ,0.001
234980_at TMEM56 transmembraneprotein56 3401 25.4 0.002
220801_s_at HAO2 hydroxyacidoxidase2(longchain) 1120 25.3 ,0.001
230573_at SGK2 Serum/glucocorticoidregulatedkinase2 495 25.3 0.0016
207958_at UGT2A1/UGT2A2 UDPglucuronosyltransferase2family,polypeptideA1/UDP 351 25.0 ,0.001
glucuronosyltransferase2family,polypeptideA2
206465_at ACSBG1 acyl-CoAsynthetasebubblegumfamilymember1 8201 24.9 0.002
208962_s_at FADS1 fattyaciddesaturase1 5240 24.8 0.003
205029_s_at FABP7 fattyacidbindingprotein7,brain 4530 24.8 0.011
239108_at FAR2 FattyacylCoAreductase2 6891 24.7 0.002
210377_at ACSM3 acyl-CoAsynthetasemedium-chainfamilymember3 591 24.5 0.003
206714_at ALOX15B arachidonate15-lipoxygenase,typeB 6853 24.5 0.003
221142_s_at PECR peroxisomaltrans-2-enoyl-CoAreductase 1668 24.5 0.0012
236175_at TRIM55 tripartitemotif-containing55 184 24.4 ,0.001
208331_at BPY2 basiccharge,Y-linked,2 1084 24.4 0.007
doi:10.1371/journal.pone.0105238.t004
accession number GSE53795. A two-component Non Negative responses as the main enriched biological processes (Figure 3a).
MatrixFactorization(NMF)model[32]wasappliedandalloweda Interestingly, the IL-17 signaling pathway and the Th17-derived
clear separation between lesional and non-lesional acne samples cytokines network were ranked fourth in the enriched GeneGo
(Figure 2).Bioinformaticanalysisofdifferentiallymodulatedgenes pathways andnetworks, respectively (Figures3band3c).
usingMetacoresoftwareidentifiedtheinflammatoryandimmune
Figure2.2-componentsNMF(NonNegativeMatrixFactorization)model.Theheatmapof904regulatedgenesonsubjects-correctedlog2-
expressions(legendcolorblueforlowexpressionandredforhighexpression).
doi:10.1371/journal.pone.0105238.g002
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Th17PathwayinAcne
Figure3.Bioinformaticanalysisofdifferentiallymodulatedgenes.Geneenrichmentanalysisinthreedifferentfunctionalontologieswere
performedusingMetaCore:biologicalprocesses(a),processnetworks(b)andcanonicalpathwaymaps(c).
doi:10.1371/journal.pone.0105238.g003
TheAffymetrixtranscriptomeresultswereconfirmedwithreal- Thus, the upregulation of Th17 pathway found with global
time PCR (RT-PCR). Among the upregulated genes were IL-23, AffymetrixanalysiswasconfirmedwithRT-PCRandalsoonthe
IL-6 and TGF-b, which all are critically involved in Th17 cell proteinlevelwithLuminexandimmunohistochemistryinpatient
activation and differentiation. Furthermore, Th17 cell products samples from Germany. Moreover, the result was similar in a
IL-17A,IL-22,IL-26,TNFandIL-17inducedchemokinesCSF2 separatepatientgroupfromFinlandinvestigatedbyRT-PCRand
and CCL20 were also expressed at higher levels in acne lesions immunohistochemistry. While IL-17A in addition to Th17 cells
(Table 5). The expression of the upregulated genes of Th17 canalsobesecretedbyotherCD3+lymphocytes,includingCD8+
lineage inducing cytokines IL-1b, IL-6, TGF-b and IL-23 were cytotoxic T-cells (Tc17) and cd T cells, the concomitantly
found significantly increased also in the Finnish cohort by RT- observed inductions of Th17-activating and Th17-related cyto-
PCR (Figure 4)as well asTh17signaling cytokineIL-17A. kines and chemokines observed in two independent patient
TheincreasedproductionofTh17relatedcytokinesintheacne populations support the observation of Th17 pathway activation
lesionswasconfirmedattheproteinlevelbycytokineprofilingof inacne lesions.
manuallycrushedskinbiopsiesfromGermanpatientsbyLuminex
technology (Table6). Furthermore, significantly higher numbers Innateimmune responses weresignificantly increased in
of IL-17A positive (IL-17A+) cells were detected by immunohis- acne lesions
tochemistry both in the papillary dermis and around sebaceous
Wewantedtoinvestigatealsoinnateimmuneresponsesinacne
folliclesinacnelesionsofFinnishpatients(Table7).IL-17A+cells
lesions. P. acnes triggers inflammatory responses via TLR2 or
were scattered in the superficial and deep layers of the dermis
Nod-like receptor 3 (NLRP3) -inflammasome, leading to the
(Figure 5a). IL-17A+ cells were mainly lymphocytes but some of
production of IL-6, and IL-8 by keratinocytes and sebocytes and
them were neutrophils and even mast cells (data not shown). IL- IL-1b, IL-12 and IL-8 by macrophages and primary monocytes,
17A+ cells were also identified in the inflammatory infiltrates in
respectively [15,16,33–35]. The expression of TLR2 and TLR4
theGermanpatientgroup(Figure5b).StronglyIL17-positivecells
weresignificantlyelevatedintheacnelesionsintheFinnishgroup
weremainlycomposedofCD3+lymphocytesinbothlesionaland
(Figure 6a). Of note, TLR2, but not TLR4, was increased in
non-lesional samples.
psoriasiscontrolsamplesasfoundearlierbyBegonetal[36].The
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Th17PathwayinAcne
n)
vs.ski
dd
ee
vv
olol
vv
FDR(innon-in ,0.001 0.08 ,0.001 0.25 ,0.001 ,0.001 ,0.001 0.31 0.004 ,0.001 0.14 0.08 0.10 ,0.001
n-
o
Foldchange(involvedvs.ninvolvedskin) 6.2 1.1 6.2 -1.4 5.6 2.4 6.2 1.1 1.5 2.4 1.3 1.3 21.6 21.8
hort. non-kin) ed
co T(ds uat
nacne RT-PCR MeanCinvolve 33 24 30 22 32 Noteval 34 30 28 31 29 34 32 34 28
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ells on-
ngtheTh17c Foldchange(involvedvs.ninvolvedskin) 1.3 1.8 4.7 1.9 4.3 2.5 1.8 4.7 1.6 2.0 3.3 1.1 1.2
zi
eri n)
ytokinescharact Affymetrixdata Meanexpression(non-involvedski 95 111 73 962 13 35 9 73 80 24 146 Notdetected 91 Notdetected 91
c
RNAlevelofmarkersand ytokines Title interleukin23,alphasubunitp19 transforminggrowthfactor,beta1 interleukin6 signaltransducerandactivatoroftranscription3 interleukin17A interleukin17F interleukin22 interleukin6 tumornecrosisfactor interleukin26 chemokine(C-Cmotif)ligand20 interleukin21 colonystimulatingfactor2 interleukin23receptor chemokine(C-Cmotif)receptor6 e;FDR,Falsediscoveryrate.
sionatthem Markersandc Genesymbol IL23 TGFB1 IL6 STAT3 IL17A IL17F IL22 IL6 TNF IL26 CCL20 IL21 CSF2 IL23R CCR6 clethresholdvaluone.0105238.t005
ExpresTable5. Th17signalling Th17activation Th17relatedcytokinesandchemokines Receptors Abbreviations:CT,cydoi:10.1371/journal.p
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Th17PathwayinAcne
Figure4.Th17relatedcytokineswereincreasedinacnelesions.Theskinbiopsieswereobtainedfromhealthyvolunteersnormalskin(NS),
fromacnepatientsnon-lesionalskin(ANS)andlesionalskin(AL)andfrompsoriasispatientsnon-lesionalskin(PNS)andlesionalskin(PL).The
expressionlevelsofcytokinesweredeterminedbyRT-PCR.TheexpressionsofinflammatorycytokinesTGF-b,IL-6,IL-1bandIL-23wereincreased
significantlybothinacneandpsoriasisbutIL-1bandTGF-bexpressionwashigherinacne.IL-23wasincreasedmoreinpsoriasislesions.IL-17Awas
significantlyexpressedinlesionalacneandpsoriasisskin.IL-22,wasslightlyelevatedinacneandpsoriasis,howeversignificantlymoreinpsoriasis.
*P,0.05,**P,0.01,***P,0.001;barsrepresentmean+SEM.RU,relativeunits;TGF-b,transforminggrowthfactor-beta;Th17,Thelpertype17.
doi:10.1371/journal.pone.0105238.g004
proinflammatory cytokines IL-1b, IL-6, TNF-a and the chemo- Th1 and T regulatory cell markers were elevated at the
kineIL-8wereallexpressedatahigherlevelinacnelesionsinthe RNA level in acne lesions
Finnishcohort(Figure4,Figure 6b).Similarly,TLR2andTLR4
In addition to the Th17 pathway, we examined other T-cell
aswellasIL-8,IL-6,andTNF-a,wereinducedinacnelesionsin subsetsinacnelesions.Mouserandco-workers[38]demonstrated
theGerman group(Table8). a subpopulation of Th1 cells generated from early inflamed acne
The expression of antimicrobial peptides (AMPs) was also lesions,butregulatoryTcells(Tregs)arenotinvestigatedinacne
increased in the acne lesions. AMPs are evolutionary conserved, lesions.IL-12isapivotalcytokineinactivatingTh1responses,and
positivelychargedmoleculesthatbindmembranesofmicrobesby isinducedinmonocytesbyP.acnesviaTLR2[6]andinvitroin
theirhydrophobicsurfacesandformporesonthemembranesand PBMCs [20]. The IL-12 subunit p40, but not p35, was found
limitthesurvivalofcommensalsandpotentialpathogensalsowith significantly increased in both acne (P,0.01) and psoriasis (P,
other mechanisms [37]. The expression of AMPs human 0.001) by RT-PCR (Figure 8). Furthermore, the Th1 polarizing
cathelicidin antimicrobial protein (hCAP18), lipocalin (LCN2), key transcription factor T-bet, Th1 effector cytokine IFN-c, and
humanbeta-defensin2(hBD2),hBD3,S100A7andS100A9were Th1 type chemokine receptor CXCR3 were also expressed at a
found significantly upregulated in acne lesions in the Finnish higher level in both acne and psoriasis lesions, indicating the
patient group by RT-PCR (Figure 7). Similar findings were involvementofTh1effectorcells(Figure 8).Similarfindingswere
observed inGerman patients(Table 9). observedintheGermancohort,sinceSTAT1apositiveregulator
PLOSONE | www.plosone.org 9 August2014 | Volume 9 | Issue 8 | e105238
Th17PathwayinAcne
Table6.ExpressionattheproteinlevelofmarkersandcytokinescharacterizingtheTh17cellsubtypeintheGermanacnecohort.
Proteinlevelpg/mg Foldchange(involved P-value(involvedvs.
Th17Signalling Symbol Title (non-involvedskin) vs.non-involvedskin) non-involvedskin)
Th17activation IL23 interleukin23,alpha 0.3 3.1 0.17
subunitp19
IL6 interleukin6 1.1 15.0 ,0.001
Th17relatedcytokines IL17A interleukin17A 1.1 3.8 0.01
andchemokines
IL17F interleukin17F 0.3 4.9 ,0.001
IL22 interleukin22 4.6 3.1 0.03
IL6 interleukin6 1.1 15.0 ,0.001
TNF transforminggrowth 1.1 2.5 0.02
factor,beta1
CCL20 chemokine(C-Cmotif) 0.4 2.6 0.04
ligand20
IL21 interleukin21 7.5 3.9 0.01
CSF2 colonystimulatingfactor2 1.1 2.2 0.01
doi:10.1371/journal.pone.0105238.t006
of Th1 development was induced, and the CXCR3 ligands by RT-PCR, suggesting that Th2 cells are not involved in the
CXCL9, CXCL10 and CXCL11, were overexpressed in lesional inflammatory response inacne andinpsoriasis (data notshown).
acne skin(Table 10).
Foxp3isaspecificmarkerofnaturalandinducedTregs,which Cellsofadaptiveimmunitywererecruitedtoacnelesions
are efficient suppressors of both innate and adaptive immune Inadditiontomeasurementofgeneexpressioninskinsamples,
responses. TGF-b and IL-10 are involved in the induction and we investigated the infiltration of key cell types in acne lesions.
function of Tregs [39]. In the Finnish cohort, the expression of Apart from IL-17A, skin biopsy tissue sections were stained for
Foxp3,TGF-bandIL-10werefoundsignificantlyup-regulatedin CD4, CD8, T-bet, Foxp3, CD68 and CD83. All sections of
acnelesionsbyRT-PCR,andthelattertwowereathigherlevels lesionalandnon-lesionalacnesamplescontainedimmunecellsin
in acne lesions compared with psoriatic lesions (Figure 8). In the papillary dermis and around sebaceous follicles. The most
German cohort, TGF-b and IL-10 displayed a slightly enhanced common cells were CD68+ macrophages and CD4+ T-cells.
expression in acne lesions and also the expression of Foxp3 was The count of CD4+ cells and CD83+ mature dendritic cells was
detected (Table 11). significantly higher both in the papillary dermis and around
Th2cellsproducedcytokinesIL-4,IL-5andIL-13wereeither follicles in acne lesions, when compared with non-lesional skin.
not detected or not modulated in the German cohort by array (Figure 5e–h, Table 7). T-bet+ cells were also detected both in
technology,anddetectedataverylowlevelintheFinnishcohort lesionalandnon-lesionalskinofacnepatients,butatsignificantly
higher numbers in acne lesions, both in the papillary dermis and
Table7. Numberofpositively stainedcells inlesionaland non-lesionalskin ofacne patients(n=16)1asassessed by
immunohistochemistry.
Positive-cellcounts2inupperdermis Positive-cellcounts2aroundfollicles
Lesional Non-lesional P-value Lesional Non-lesional P-value
CD4 33(22–52.8) 16.5(9.3–26.3) 0.007 52(44–127.5) 8(4–14.5) 6.561025
CD8 6(2.3–15.3) 3(1.3–9.5) 0.225 11(4.5–18.5) 1(1–2) 8.561025
T-bet 6(3–17) 2.5(1–3) 0.005 16.5(3.5–31.5) 0(0–1.8) 8.661025
IL–17A 4.5(2.3–8) 2(2–3) 0.030 6(3.5–9.3) 3(1.3–5.3) 0.023
Foxp3 4.5(2–9.8) 1(1–3) 0.003 6(0.8–13) 1(1–2) 0.072
CD68 38(18.3–68) 5.5(1–13) 3.1610–5 77.5(27–131.5) 3.5(2.3–5.8) 2.661025
CD83 7(2.3–13) 1.5(0.3–2.8) 1.5610–4 12(4.5–27) 1(0–2) 2.361026
Abbreviations:IHC,immunohistochemistry;IQR,interquartilerange.
116oftotal20acnepatientssamplesinFinnishpatientcohortwereexaminedbyIHC.
2Positive-cellcountsperfieldimageareexpressedasmedians(IQR).StatisticallysignificantdifferenceofP,0.05inthelesionalacneskincomparedtonon-lesionalskin.
SignificantPvaluesarebold.
doi:10.1371/journal.pone.0105238.t007
PLOSONE | www.plosone.org 10 August2014 | Volume 9 | Issue 8 | e105238