Production of Superoxide Anions by Keratinocytes Initiates P. acnes-Induced Inflammation of the Skin

14 Pages·2009·0.88 MB·English

Production of Superoxide Anions by Keratinocytes
Initiates P.acnes-Induced Inflammation of the Skin
Philippe A. Grange1, Christiane Che´reau2,3, Joe¨l Raingeaud4, Carole Nicco2, Bernard Weill2, Nicolas
Dupin1,5., Fre´de´ric Batteux2,3.*
1LaboratoiredeRechercheenDermatologie,EA1833,Faculte´deMe´decine,Universite´ParisDescartes,Paris,France,2Laboratoired’ImmunologieEA1833,Faculte´de
Me´decine,Universite´ParisDescartes,Paris,France,3ERTi«Plateformed’e´tudedustressoxydantenoncologieetdanslesmaladiesinflammatoires»,Faculte´deMe´decine,
Universite´ParisDescartes,Paris,France,4INSERMU749,Universite´Paris-sud,Faculte´dePharmacie,Chatenay-Malabry,France,5ServicedeDermatologie-Ve´ne´re´ologie,
HoˆpitalCochin–PavillonTarnier,AP-HP,Paris,France
Abstract
Acne vulgaris is a chronic inflammatory disorder of the sebaceous follicles. Propionibacterium acnes (P. acnes), a gram-
positive anareobic bacterium, plays a critical role in the development of these inflammatory lesions. This study aimed at
determiningwhetherreactiveoxygenspecies(ROS)areproducedbykeratinocytesuponP.acnesinfection,dissectingthe
mechanism of this production, and investigating how this phenomenon integrates in the general inflammatory response
induced by P. acnes. In our hands, ROS, and especially superoxide anions (O N2), were rapidly produced by keratinocytes
2
uponstimulationbyP.acnessurfaceproteins.InP.acnes-stimulatedkeratinocytes,O N2wasproducedbyNAD(P)Hoxidase
2
through activation of the scavenger receptor CD36. O N2 was dismuted by superoxide dismutase to form hydrogen
2
peroxidewhichwasfurtherdetoxifiedintowaterbytheGSH/GPxsystem.Inaddition,P.acnes-inducedO N2abrogatedP.
2
acnes growth and was involved in keratinocyte lysis through the combination of O N2 with nitric oxide to form
2
peroxynitrites.Finally,retinoicacidderivates,themostefficientanti-acneicdrugs,preventO N2production,IL-8releaseand
2
keratinocyte apoptosis, suggesting therelevance ofthis pathway inhumans.
Citation: Grange PA, Che´reau C, Raingeaud J, Nicco C, Weill B, et al. (2009) Production of Superoxide Anions by Keratinocytes Initiates P. acnes-Induced
InflammationoftheSkin.PLoSPathog5(7):e1000527.doi:10.1371/journal.ppat.1000527
Editor:AmbroseCheung,DartmouthMedicalSchool,UnitedStatesofAmerica
ReceivedFebruary6,2009;AcceptedJuly1,2009;PublishedJuly24,2009
Copyright: (cid:1)2009 Grangeet al.Thisisanopen-access articledistributedunder thetermsof theCreativeCommonsAttribution License, which permits
unrestricteduse,distribution,andreproductioninanymedium,providedtheoriginalauthorandsourcearecredited.
Funding:Thisworkhasbeensupportedby‘‘AssociationdeRechercheenVirologieetDermatologie’’(ARVD).
CompetingInterests:Theauthorshavedeclaredthatnocompetinginterestsexist.
*E-mail:[email protected]
.Theseauthorscontributedequallytothiswork.
Introduction reactive oxygen species (ROS) that can damage the follicular
epithelium.
Acne vulgaris is a chronic inflammatory disorder of the Besidetheimmuneresponseofthehost,adirecteffectofP.acnes
sebaceous follicles. Acne is the most common skin disease, on keratinocytes has also been suspected in the initiation of the
estimated to affect up to 80% of individuals at some point inflammatory process. Indeed, P. acnes interacts with toll-like
between the ages of 11 and 30 years. Despite its common receptorsTLR-2andTLR-4onkeratinocytes[3].Thisinteraction
occurrence, the pathogenesis of acne is not fully understood. induces the release of inflammatory cytokines such as IL-1a, IL-
Excessive shedding of epithelial cells from the walls of follicles 1b,IL-8,GM-CSF,andTNF-a[4,5].Althoughnothingisknown
combined with increased amounts of sebum produced by about the interaction between P. acnes or any other bacteria with
associated sebaceous glands are two important factors that keratinocytes in terms of reactive oxygen species (ROS) produc-
contribute to follicular obstruction. This obstruction leads to the tion, purified tuberculine has been shown to activate TLR-2 on
formationofmicrocomedos,whicharebelievedtoprecedelesions keratinocytes, leading to the production of ROS during tubercu-
of acne. These microcomedos may evolve into clinically visible losis infection [6]. In addition, Vitreoscilla filiformis has been
comedos and/orinflammatory lesions. identified to activate MnSOD as an inducible free-radical
Propionibacterium acnes (P. acnes), a gram-positive anaerobic scavenger in keratinocytes [7]. Furthermore, keratinocytes are
bacterium part of the normal skin flora, plays a critical role in knowntoproduceROSuponexposuretotoxiccompoundssuch
the development of inflammatory lesions in acne [1]. Various as inorganic arsenic [8] or to ultraviolet radiations [9,10].
mechanismscanexplaintheroleofP.acnesinskininflammation. Whatever the mechanism implicated in the induction of skin
First, it is widely accepted that inflammation may be induced by inflammation by P. acnes, ROS are probably involved in that
the immune response of the host to P. acnes. Chemotactic process since the production of hydrogen peroxide (H O ) is
2 2
substances released from the bacteria attract polymorphonuclear increased in neutrophils from acne patients [11]. Moreover, the
leukocytes to the site of inflammation. Those cells are activated decrease in superoxide dismutase (SOD) activity in patients with
locallytoproduceinflammatorycytokinessuchasTNF-a,IL-1b, acne lesions [12]iscorrelated with theseverity of acne[13].
and IL-8 [2]. After phagocytosis of the bacteria, the attracted ROS are short-lived small molecular structures that are
neutrophilsarethoughttoreleaselysosomalenzymesandproduce continuously generated at low levels during the course of normal
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P.acnes-InducedROSProductioninKeratinocytes
of ROS, and especially of O N2, is a very early event occurring
Author Summary 2
almost immediately after the stimulation of keratinocytes with P.
Acne vulgaris is a chronic inflammatory disorder of the acnes. The production of O N2 by HaCat keratinocytes was
2
sebaceous follicles. It is the most common skin disease, identicalwhetherthecellshadbeenstimulatedbyanextractofP.
affectingupto80%ofindividualsatsomepointbetween acnes surface proteins or by the whole bacteria. O N2 production
2
the ages of 11 and 30 years. Propionibacterium acnes (P. was measured using DHE, and cell death estimated using YO-
acnes) plays a role in the development of inflammatory
PRO-1 were dose-dependent (Figure3).
acne lesions, but whether it causes inflammation by itself
orthroughindirectmechanismsisnotclearyet.Therefore,
OriginofROSproducedbykeratinocytesstimulatedwith
byexposingepidermalcellstoP.acnesinvitro,wetested
whetherreactiveoxygenspecies(ROS)production(oxida- P. acnes
tive burst) was involved in the inflammatory process. We Superoxide anions can originate from the mitochondrial
found that one particular ROS, superoxide anion, was complex I or III of the respiratory chain, or from the cytosolic
generated by epidermal cells following P. acnes stimula- enzymesNAD(P)Hoxidaseor xanthineoxidase.Incubation ofP.
tion. This phenomenon is associated with the production
of a soluble pro inflammatory molecule, IL-8, and
epidermal cell death. The abrogation of P. acnes-induced
oxidative burst by the most commonly used and most
efficient treatments of acne suggests that superoxide
anions produced by epidermal cells are critical in the
development ofacne inflammatory lesions.
aerobic metabolism. They are also part of the inflammatory
processthataimsatkillingoreliminatinginvasivemicroorganisms
and/or eliminating damaged tissular structures. Among the large
number of ROS that have been described, superoxide anion
(O N2) and hydrogen peroxyde (H O ) play prominent roles. On
2 2 2
the other hand, the interaction between O N2 and nitric oxide
2
(NO), leads to the formation of highly reactive peroxynitrites
(ONOON2). ROS interact strongly with a variety of molecules
including lipids, proteins, and nucleic acids. Produced in large
amounts, ROS can lead to apoptotic or necrotic cell death. To
counteract the overproduction of ROS, skin is equipped with
antioxidant mechanisms including anti-oxidant enzymes such as
superoxide dismutase (SOD) that detoxifies O N2, catalase, and
2
glutathioneperoxidase(GpX)thatusesreducedglutathione(GSH)
todetoxify H O intowater [14].
2 2
Inthiswork,wehaveusedaninvitromodeltoinvestigateROS
production by keratinocytes upon P. acnes stimulation. We have
dissected the control mechanisms of this production, and
investigated how they fit into the general inflammatory response
induced by P.acnes.
Results
ROS production by P. acnes-stimulated keratinocytes is
dose-and time-dependent
P.acnesincreasedtheproductionofO N2,NOandH O bythe
2 2 2
immortalized keratinocyte cell line HaCaT in a dose-dependent
manner (Figure 1A, B and C). At the highest concentration of P.
acnes, O N2, NO and H O levels were increased by 85%
2 2 2
(P,0.05), 44.5% (P,0.05) and 41% (P,0.05), respectively. We
then evaluated the kinetics of ROS production (Figure 2). The
production of O N2, was significantly increased 15min after P.
2
acnes stimulation (P,0.05). The production reached its peak one
hourafterthestimulation,thenprogressivelydeclined(Figure2A).
Incontrast,bothNOandH O productionsincreasedslowlyand
2 2
reached their highest levels after 24h of incubation with P. acnes Figure1.ROSproductionbyP.acnes-stimulatedkeratinocytes.
(Figures2BandC).SincekeratinocytesstimulatedbyP.acnescan HaCaTcellswereincubatedfor18hwithP.acnesatanMOIof50,5,0.5,
produce IL-8 [3], we next compared the kinetics of ROS and 0.05 (gray bars). Control experiments were done on HaCaT cells
alone (black bars). Measurement of (A) superoxide anion, (B) nitric
productionwiththatofIL-8productionuponP.acnesstimulation
oxide,and(C)hydrogenperoxidewasrealizedbyspectrofluorometryas
(Figure 2D). Significant levels of IL-8 protein appeared 2 h after
described in Materials and Methods. Data are means6SD of two
incubation with P. acnes (P,0.05) and increased along with ROS separateexperiments.
production. Altogether, these results indicate that the production doi:10.1371/journal.ppat.1000527.g001
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P.acnes-InducedROSProductioninKeratinocytes
Figure 3. Superoxide anion production by keratinocytes
stimulated withP.acnestotal surface proteins extract. HaCaT
cellswereincubatedfor18hinthepresenceofseveralP.acnessurface
proteins whose concentrations ranged from 0.08 to 2.4mg/ml (gray
bars).ControlexperimentsweredoneonHaCaTcellsinpresenceofPBS
(graybars).Measurements of(A)superoxideanion,and (B)celldeath
were performed by spectrofluorometry with DHE and YO-PRO-1,
respectively as described in Materials and Methods. The amount of
cellspresentinwellsafter18hofincubationwas66,73,77,85,89,and
98%,respectively.Dataaremeans6SDoftwoseparateexperiments.
doi:10.1371/journal.ppat.1000527.g003
acnes-stimulated keratinocytes with rotenone and antimycin that
inhibit the mitochondrial respiratory chain complexes I and III,
respectively, did not significantly alter the production of O N2
2
(Figure 4A). Incubation of P. acnes-stimulated keratinocytes with
DPI (a NAD(P)H oxidase inhibitor) significantly decreased O N2
2
production (P,0.03), while incubation with allopurinol (a
xanthineoxidaseinhibitor)hadnoeffect(Figure4A).Toconfirm
thatNoxisthemainsourceofO N2inkeratinocytesstimulatedby
2
P. acnes, the level of Nox1 was knocked down using RNA
interference. The small interfering RNA (siRNA) Nox1A-siRNA
wasusedasdescribedpreviously[15].Nox1A-siRNAdramatically
decreased the production of O N2 upon stimultion by P. acnes in
2
transfected-keratinocytes,withnearly100%inhibitionafter3 hof
stimulation (Figure 4C). Keratinocytes treated with scrambled
sequence siRNA produced similar levels of O N2 as non-
2
transfected cells. These results demonstrated that O N2 is mainly
2
produced by NAD(P)H oxidase in P. acnes-stimulated keratino-
cytes.
Figure 2. Kinetics of ROS versus IL-8 production in P.acnes-
stimulatedkeratinocytes.HaCaTcellswereincubatedwithP.acnes ROS detoxification pathways involved in P. acnes-
(MOIof50)foraperiodoftimerangingfrom0.25to24h.(A)Superoxide stimulated keratinocytes
anion,(B)nitricoxide,(C)hydrogenperoxidelevelsweredeterminedby
In order to determine the pathways implicated in the
spectrofluorometry as described in Materials and Methods. (D) IL-8
detoxification of ROS produced by P. acnes-stimulated keratino-
production was determined by ELISA as described in Materials and
Methods.Dataaremeans6SDoftwoseparateexperiments. cytes, we used specific modulators of the enzymatic systems
doi:10.1371/journal.ppat.1000527.g002 involved in ROS metabolism. Superoxide anions are converted
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P.acnes-InducedROSProductioninKeratinocytes
(Figure 4A). Hydrogen peroxide is converted into H O by two
2
setsofenzymes,catalaseandtheGSH/GPxsystem.Theelevation
ofhydrogenperoxidelevelscanbecausedeitherbyanincreasein
superoxide dismutation as observed following incubation with
MnTBAP(P,0.004)orCuDIPS(P,0.02)orbyadecreaseinthe
detoxificationpathways(Figure4B).Specificinhibitionofcatalase
by aminotriazol (ATZ)oraddition of exogenous catalase, hadno
effect on the levels of hydrogen peroxide (Figure 4B). Thus, the
catalase pathway is not involved in the control of hydrogen
peroxidedetoxificationinoursystem.DepletingGSHwithBSO,
inhibited GPx and significantly increased H O production
2 2
(P,0.05), while adding exogenous GSH or its precursor NAC,
significantly decreased H O levels (P,0.004 and P,0.01,
2 2
respectively) (Figure 4B). Those results highlight the role of
GSH/GPx in keratinocytes to counteract the overproduction of
ROSinducedbyP.acnes:superoxideanionsaredismutedbySOD
into hydrogen peroxide, which is further detoxified into H O
2
through theGSH/GPx pathway.
ROS toxicity and production of nitrosyl residues by P.
acnes-stimulated keratinocytes
Given the high toxicity of ROS, we were prompted to
investigate if the levels of O N2 produced by keratinocytes could
2
impactcellularviability.Theapoptosisofkeratinocytesinducedby
P. acnes alone was estimated by YO-PRO-1 (Figure 5A) and
TUNELstaining(Figure5B).Inordertodeterminethenatureof
ROSinvolvedinP.acnes-inducedcellulartoxicity,wepre-treated
keratinocytes wih specific modulators of the enzymes involved in
the production of O N2 and H O and measured the death of
2 2 2
keratinocytes upon stimulation with P. acnes. Inhibition of
superoxide anions by allopurinol, DPI or MnTBAP, significantly
decreased P. acnes-induced keratinocyte apoptosis (P,0.005,
P,0.005, P,0.001, respectively), whereas DDC and antimycin,
two compounds that increase O N2 production, increased cell
2
death(P,0.007andP,0.01,respectively)(Figure5A).CuDIPS,a
mimicofthecytosolicsuperoxidedismutase,increasedcelldeath.
This is explained by the cytotoxic properties of this molecule on
the cells. No major effect was observed on the rate of cell death
withmoleculesmodulatingH O production,suchasATZ,BSO,
2 2
NAC, GSHorcatalase.
PeroxinitritesresultfromthecombinationofO N2andNO.They
2
Figure 4. ROS detoxification pathways involved in P.acnes- are highly reactive metabolites that create nitrosyl residues on
stimulated keratinocytes. (A) Superoxide anions and (B) hydrogen proteins and alter their functions. Therefore, the levels of nitrosyl
peroxideproductionsbyHaCaTcellsweredeterminedafterincubation residuesnotonlyreflecttheintensityoftheoxidativeattackbutare
for18hwithP.acnes(MOIof50)alone(blackbars)orwithP.acnesin
alsomarkersofthecellulardamagescreatedbytheoxidativeburst.
thepresenceofspecificmodulatorsofenzymaticsystemsinvolvedin
As shown by flow cytometry, 3-nitrosotyrosyl residues were dose-
ROS metabolism (gray bars). The concentrations used were the
following: 40mM rotenone, 40mM antimycin, 40mM allopurinol, dependentlyincreasedfromwithverylowconcentrationsofP.acnes
40mMDPI,2mMDDC,400mMATZ,0.8mMBSO,100mMmanganese (Figure6A).Thisresultisinagreementwiththeobservationthatthe
[III] tetrakis (5,10,15,20)-benzoic acid porphyrin (MnTBAP), 400mM nitricoxidesynthase(NOS)isactivatedinkeratinocytesstimulated
copper[II]diiosopropylsalicylate (CuDIPS), 3.2mM N-acetylcysteine withP.acnes.Theexpression ofiNOSwassteadyinkeratinocytes
(NAC), 1.6mM GSH, 20U catalase. (C) HaCaT cells were pretreated
during a period of time of 24h as determined by RT-PCR
with the Nox1A-siRNA sequence or with a scrambled sequence as
(Figure 6B) and by RT-qPCR (data not shown). This data is
describedinMaterialsandMethods.Superoxideanionsproductionwas
measuredafterstimulationbyP.acnes(MOIof50)inuntreatedHaCaT consistentwiththosepresentedinFigure1,showingthatNOwas
(black bar) or in siRNA pretreated HaCaT (gray bars). ROS production produced early after P. acnes incubation, making its interaction
was measured by spectrofluorometry as described in Materials and between O N2 and NO possible. Altogether, those experiments
2
Methods.Dataaremeans6DSoftwoseparateexperiments. suggested that the toxicity of O N2 produced by keratinocytes
doi:10.1371/journal.ppat.1000527.g004 2
stimulatedwithP.acneswasdependentonthecombinationwithNO
andtheproductionofnitrosylresidues.
into hydrogen peroxide by SOD. Inhibiting SOD by the specific
inhibitorDDCsignificantlyincreasedO N2productionbyP.acnes- P. acnes-induced O N2 production controls IL-8 levels
2 2
stimulated keratinocytes (P,0.003) (Figure 4A). By contrast, InordertoevaluatetheroleofO N2intheproductionofIL-8
2
incubation of keratinocytes with MnTBAP or CuDIPS, two byP.acnes-stimulatedkeratinocytes,wemeasuredthelevelsofIL-8
SODmimics,significantlydecreasedO N2productionbyP.acnes- produced in presence of the various ROS modulators (Figure 7).
2
stimulated keratinocytes (P,0.05 and P,0.04, respectively) All the molecules that inhibited O N2 production also decreased
2
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P.acnes-InducedROSProductioninKeratinocytes
Figure5.RelationshipbetweenP.acnes-inducedO N2productionandkeratinocyteapoptosis.(A)HaCaTcellswereincubatedfor18hwith
2
P.acnes(MOIof50)(blackbars)orwithP.acnesinthepresenceofmodulatorsofROSproduction(graybars).Theconcentrationsusedwere40mM
rotenone,40mMantimycin,40mMallopurinol,40mMDPI,2mMDDC,400mMATZ,0.8mMBSO,100mMmanganese[III]tetrakis(5,10,15,20)-benzoic
acidporphyrin(MnTBAP),400mMcopper[II]diiosopropylsalicylate(CuDIPS),3.2mMN-acetylcysteine(NAC),1.6mMGSH,20Ucatalase.Celldeathwas
assessedspectrofluorometricallyasdescribedinMaterialsandMethodsandexpressedasmeans6SDfromtwoexperimentscarriedoutinduplicates.(B)
InductionofDNAfragmentationinHaCaTcellsfollowingincubationwithP. acneswasassessedbyTUNELstainingasdescribedinMaterialsand
Methods.Panels1and2correspondtoHaCaTcellsalone.Panels3and4correspondtoHaCaTcellsincubatedwithP.acnesfor18hatanMOIof50.
doi:10.1371/journal.ppat.1000527.g005
IL-8 synthesis, but only the decrease induced by DPI reached implicated in the recognition of P. acnes. P. acnes-stimulated
statisticalsignificance(P,0.03).Ifallthemoleculesthatincreased keratinocyteswereincubatedwithantibodiestoTLR-2orCD36,
O N2 levels also increased IL-8 production, only the massive andIL-8andO N2productionsmeasured(Figure8).Theantibody
2 2
increasecausedbyDDCreachedsignificance(P,0.04)(Figure7). directed to TLR-2 was known as a blocking agent for the
Both ATZ and NAC significantly decreased IL-8 production. production of IL-8 by keratinocytes after stimulation by P. acnes
Since it has previously been shown that ATZ has no effect on [3]. This was confirmed by the reduction in IL-8 production by
H O productionwhileNACandGSHdo(Figure4),theseresults 65% (P=0.01) (Figure 8A), whereas no change was observed in
2 2
suggestthattheeffectsofATZandNAConIL-8productionare O N2 production (Figure 8B). However, when P. acnes-stimulated
2
independentoftheregulationofH2O2andaremorelikelylinked keratinocytes were incubated in the presence of the antibody to
to intrinsic properties of those products. Altogether these results CD36,theproductionofO N2wasreducedby51%(P=0.03)and
2
suggest that the toxicity of ROS on P. acnes-stimulated keratino- theproduction of IL-8was completely abolished.
cytes ismainly caused by O N2 which also exerts a positive effect
2
on IL-8 production. Effect of ROS on P. acnes growth
Wefirstcompared the relative sensitivity of HaCaT cells andP.
RoleofthescavengerreceptorCD36intheproductionof acnes to the toxic effect of O N2. HaCaT cells and P. acnes were
2
superoxyde anions incubatedseparatelywithasolutioncontainingO N2.Thegrowthof
2
Since O N2 elicits IL-8 production by keratinocytes stimulated P. acneswas dose dependently inhibited by O N2 while the HaCaT
2 2
with P. acnes, we investigated which surface proteins could be cells appear to be more resistant than P. acnes at the same O N2
2
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P.acnes-InducedROSProductioninKeratinocytes
Figure6.NitrosylresidueformationandiNOSexpressioninP.acnes-stimulatedkeratinocytes.HaCaTcellswereincubatedfor18hwithP.
acnes(A =0.2and1.0)andharvestedfollowingtrypsinetreatmentafterremovalofbacteria.(A)Cellswereincubatedwithprimarymousemonoclonal
600nm
antibodytonitrotyrosineorwithanti-CD8monoclonalantibodyascontrol.Boundantibodiesweredetectedusingagoatanti-mouseIgG-FITCsecondary
antibody.CellswerethenanalyzedbyflowcytometryasdescribedinMaterialsandMethods.(B)TotalRNAwasextractedandtheiNOSandGAPDHmRNA
expressionwasanalysedbyRT-PCR.PCRfragmentswerevisualizedunderU.V.ona1.7%agarosegelafterstainingwithethidiumbromide(1mg/ml).
doi:10.1371/journal.ppat.1000527.g006
concentration (Figure 9A). We then tested the hypothesis that the inthepresenceofZnSO ,doxycycline,nicotinamide,nitroimida-
4
ROS produced by keratinocytes, and particularly O N2, could be zol, retinol, retinoic acid, or isotretinoin (Figure 10). The
2
responsible for the inhibition of the growth of P. acnes (Figure 9B). production of superoxide anions was reduced by all the drugs
When P. acnes-stimulated keratinocytes were preincubated with tested, at least at the highest concentration (0.05%), except for
MnTBAP,aMnSODmimicthatdetoxifiesO N2,orwithDPIthat nicotinamide (Figure 10A). IL-8 production was reduced neither
2
inhibitsNAD(P)Hoxydase,thegrowthofthebacteriawasrestored. byZnSO4atlowconcentration(0.01%)norbynicotinamide,but
Reciprocally,whenkeratinocyteswherepreincubatedwithDDC,a all the others drugs tested were effective. This is particularly the
SODinhibitor,thebacterialgrowthwasdecreased. case for retinoic acid derivates that completely abolished IL-8
production (Figure 10B). The percentage of cells present in the
wellsafterincubationrangedfrom68to91%(FigureS1).Allthe
Anti-acne drugs inhibit O N2 production, IL-8 synthesis
2 drugs except ZnSO4 and nicotinamide reduced the apoptosis of
and keratinocyte apoptosis keratinocytes stimulated by P. acnes at least at the highest
Inordertoevaluatetheeffectsofthemostcommondrugsused concentration tested (0.05%). This is particularly the case for
inthetreatmentofacne,HaCaTcellswerestimulatedbyP.acnes retinoicacidderivates(P,0.03inallcases)andfortheantibiotics
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P.acnes-InducedROSProductioninKeratinocytes
Figure7.RelationshipbetweenROSandIL-8productionsinP.acnes-stimulatedkeratinocytes.HaCaTcellswereincubatedfor18hwith
P.acnesalone(MOIof50)(blackbar)orwithP.acnesinthepresenceofROSmodulators(graybars).Theconcentrationsusedwere40mMrotenone,
40mM antimycin, 40mM allopurinol, 40mM DPI, 2mM DDC, 400mM ATZ, 0.8mM BSO, 100mM manganese[III]tetrakis(5,10,15,20)-benzoic acid
porphyrin(MnTBAP),400mMcopper[II]diiosopropylsalicylate(CuDIPS),3.2mMN-acetylcysteine(NAC),1.6mMGSH,20Ucatalase.IL-8concentration
wasmeasuredinculturesupernatantsbyELISAasdescribedinMaterialsandMethods.Dataaremeans6SDoftwoseparateexperiments.
doi:10.1371/journal.ppat.1000527.g007
doxycycline (P,0.02) and nitroimidazole (P,0.03) (Figure 10C). suggest that the anti-acne drugs are active on the production of
The rate of cell death in the presence of the various compounds O N2 and IL-8 as well as on the decrease in the death rate of
2
alonerangedfrom0to26%(FigureS2).Altogether,theseresults keratinocytes.
Discussion
This report describes the production of ROS by keratinocytes
uponbacterialinfectionbyP.acnes.Theproductionofsuperoxide
anionstakesplaceatleastonehourpriortothatofnitrixoxideand
hydrogen peroxide. The same kinetics is observed following UV
radiation or arsenite intoxication[8].
Superoxide anions can originate from the cytosolic enzymes
NAD(P)H oxidase, or xanthine oxidase, or from the complexes I
orIIIofthemitochondrialrespiratorychain.TheuseofDPI,an
inhibitor of NAD(P)H oxidase and more specifically knocking
down Nox1 by small RNA interference clearly shows that, in P.
acnes-stimulated keratinocytes, O N2 is produced by NAD(P)H
2
oxidase. This data is in line with a recent report showing that
NAD(P)H oxidase is the major source of UVA-induced ROS in
human keratinocytes where mitochondria are rapidly damaged
after UVB exposure [16]. However, to date, no link between a
specific damage of the mitochondrial respiratory chain and the
production of O N2 has been established [15]. Under our
2
experimental conditions, superoxide anions are dismuted by
superoxide dismutase to form H O , which is further detoxified
2 2
into water by the GSH/GPx system and not by the catalase
pathway. In contrast, H O generated by UVB applied to
2 2
keratinocytes is detoxified through both the catalase and the
Figure8.P.acnesinducestheproductionofsuperoxideanions
GPx pathways. Usually, catalase finely tunes down H O levels,
viaCD36.HaCaTcellswerepretreated2hwithhumananti-TLR-2or 2 2
while the glutathione system (GPx and reduced glutathione) is
human anti-CD36 monoclonal antibodies (black bars), goat anti-IgG
antibody (white bar) and incubated 3h with P. acnes (A =1.0). more specialized in buffering acute oxidative stress. This is
600nm
ControlexperimentswereruninparallelwithP.acnesalone(graybar). probably whathappens inthecaseof P.acnesinfection.
(A)IL-8concentrationsweremeasuredinculturesupernatantsbyELISA, However, the key-element for P. acnes-induced apoptosis of
asdescribedinMaterialsandMethodsandareshowedminusthevalue keratinocytes is O N2 and not H O . O N2 can be toxic per se or
obtained with the HaCaT cells treated with the mAb alone. (B) O N2 2 2 2 2
2 following its combination with NO to form peroxynitrites
productionwasmeasuredspectrofluometricallyoveraperiodof3has
describedinMaterialsandMethods.Dataarepresentedasmeans6SD (ONOON2), a phenomenon that requires the activation of
ofthreeseparateexperiments. inducible nitric oxide synthase (iNOS). We confirm that P. acnes
doi:10.1371/journal.ppat.1000527.g008 induces the formation of nitrotyrosine residues on proteins, a
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P.acnes-InducedROSProductioninKeratinocytes
Figure9.EffectofROSonP.acnesgrowth.(A)HaCaTcells(darkline)andP.acnessuspension(Abs =0.5)(grayline)wereincubatedfor2h
600nm
underappropriateconditionswithvariousconcentrationsofchemicallygeneratedO2N2rangingfrom0,0191mMto10mM.TheviabilityofHaCaT
cellswasdeterminedbytheMTTassayasdescribedinMaterialsandMethods.P.acnessuspensionwasthenincubatedfor5daysat37uCunder
anaerobicconditionsandthebacterialgrowthwasevaluatedbyculturingbacteriaonRCMsolidmediaandbymeasuringtheAbsat600nm.(B)
HaCaTcellswerepreincubatedwith40mMDPI,2mMDDC,50mMMnTBAP,andstimulatedwithP.acnes(MOIof50)inDMEM10%SVFwithout
antibioticsat37uC,5%CO2.After5hofstimulation,liquidRCMwasaddedtoeachwellandtheincubationfor5daysat37uCunderanaerobic
conditionswasstarted.P.acnesgrowthwasthenevaluatedbymeasuringtheAbsat600nmandbyculturingbacteriaonRCMsolidmedia.Control
experimentwasruninparallelwiththeP.acnesinDMEM10%SVFwithoutantibioticsalone.
doi:10.1371/journal.ppat.1000527.g009
footprint of in vivo peroxinitrite production [17]. Similarly, Keratinocytes are the first line of defense against external
keratinocytes exposed to UVB or arsenite produce both O N2 aggressions; they participate in the innate immune response by
2
and NO, potentially leading to peroxinitrite formation [8,18]. In secreting soluble factors with chemotactic activity for leukocytes
our model, the production of NO by P. acnes-stimulated and neutrophils. Thus, P. acnes triggers the secretion of IL-1a,
keratinocytes is correlated with the steady expression of iNOS, TNF-a [4], and the chemokine IL-8 [3] which have been
asalreadyobservedinkeratinocytes[18].Thosedatasuggestthat implicated in the inflammatory process of acne. Using activators
the cytotoxicity mediated by ROS in our model involves the and inhibitors of the O N2 production, we have been able to
2
overproduction of O N2 and also the nitrosylation of amino acid modulate the production of IL-8 upon stimulation by P. acnes.
2
residues onproteins. Particularly,DPIaninhibitoroftheNADPHoxidase,significantly
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P.acnes-InducedROSProductioninKeratinocytes
Figure10.Effectofanti-acnetreatmentsonO N2andIL-8productioninP.acnes-stimulatedkeratinocytes.HaCaTcellswereuntreated
2
(blackbar)orincubatedfor18hwithP.acnes(MOIof50)alone(whitebar)orwithP.acnesinthepresenceofnicotinamide,zincsulfate,doxyciclyne,
nitroimidazole, retinol, retinoic acid, and isotretinoin at 0.01% (gray bars) or 0.05% (white bars). (A) O N2 production was determined
2
spectrofluorometricallywithDHE,(B)IL-8concentrationwasmeasuredbyELISA,(C)andcelldeathwasassessedspectrofluorometricallyusingYO-
PRO-1asdescribedinMaterialsandMethods.Dataaremeans6SDoftwoseparateexperiments.
doi:10.1371/journal.ppat.1000527.g010
decreases IL-8 production, whereas DDC, a SOD inhibitor that with a monoclonal antibody decreases the production of IL-8 as
increases O N2 levels, dramatically increases IL-8 production by describedpreviously[3],ithasnoeffectonO N2production.We
2 2
keratinocytes. The question was then to determine the pathway also tested the role of CD36, a scavenger molecule expressed on
through which P.acnes stimulates keratinocytes. Several previous keratinocytes [24]. The generation of ROS by the NAD(P)H
observations suggested the implication of the Toll-like receptor oxidase-NOX system has already been observed following the
(TLR) pathway. TLRs can recognize conserved molecular activationofscavengerreceptorsinvitro[25]andinvivoinamurine
structuresatthesurfaceofbacteria.TLR-2,presentatthesurface model of cerebral ischemia [26]. This receptor is a sensor of
ofkeratinocytes[19,20],isupregulatedinacnelesions[21]andis microbial diacylglycerides that signals via the TLR-2/6 heterodi-
potentially involved in the recognition of P. acnes during the mer. In response to bacterial lipoteichoic acid (LTA) and
inflammatory process [22]. Moreover, P. acnes-stimulated TLR-2 diacylated lipoproteins, CD36 associates with TLR-2/6 [24,27].
inducesIL-8releasebykeratinocytes[3,23].Wehaveobserveda Although it does not express LTA, P. acnes expresses a closely
time-lagbetweentheearlyproductionofO N2andthesecretionof related amphiphilic antigen, a lipoglycan containing mannosyl,
2
IL-8 that occurs 2 h later, that probably corresponds to the glucosyl, galactosyl residues, and an amino sugar, diaminohex-
activationoftheTLR-signalingmediatedpathway.Therefore,we uronic acid [28,29]. We observed that, blocking CD36 with a
hypothesized that the molecular mechanism responsible for O N2 monoclonal anti-CD36 antibody in P. acnes-stimulated keratino-
2
productionisTLR-independent.Indeed,whereasblockingTLR-2 cytes,significantlydecreasesboththelevelofO N2andthatofIL-
2
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P.acnes-InducedROSProductioninKeratinocytes
Figure 11. Proposed molecular mechanisms through which P.acnesinduces ROS and IL-8 production in keratinocytes. Surface
componentsofP.acnesarerecognizedbybothCD36andTLR-2.CD36triggerstheproductionofO N2throughtheNADPHoxidasepathway(NOX)
2
and combines with NO to form peroxinitrites which, in turn, activate p38 and ERK MAPKs, thus contributing to IL-8 production. In parallel, IL-8
productionisactivatedthroughtheTLR-2signallingpathway.
doi:10.1371/journal.ppat.1000527.g011
8. In our model, IL-8 secretion is triggered by the binding of P. neuroblastomacelllineanddecreaseTPA-inducedO N2produc-
2
acnes to TLR-2 and modulated by the generation of superoxide tioninmousekeratinocytes[34].Inconclusion,keratinocytesare
anions resulting from the binding of P. acnes to CD36. In not mere targets of the innate immune response but are directly
phagocytic cells, Nox1 oxidizes NADPH on the cytosolic side of involved in the defence mechanisms aiming at eliminating
thecellular membraneandreducesoxygenacrossthemembrane pathogens. In response to P. acnes, keratinocytes can produce
to generate O N2 which contributes to the killing of P. acnes [30]. massiveamountsofROSthat,inreturn,inhibitbacterialgrowth.
2
On the other hand, in keratinocytes, Nox1 is localized in the Those ROS do not only eliminate the bacteria but also generate
nucleus [31] and could release O N2 into the cytoplasm. inflammation. Thus, we hypothesize that the severity of acne
2
Therefore, we hypothesized that nuclear Nox1 could generate dependsonthebalancebetweentheabilityoftheP.acnesstrainto
O N2 which combine with steadily NO to form peroxinitrites. induceapotentimmuneresponse[3]andthecapabilityofthehost
2
Peroxinitrites activate p38 and ERK in the MAPK pathways, togenerateandtodetoxifytheROSproduced[11,13].Therefore,
contributing to the tight regulation of IL-8 production by O N2 inhibiting this inflammatory reaction using appropriate antioxi-
2
[32,33] (Figure 11). In addition, O N2 produced by keratinocytes dant molecules could be considered as a potential treatment of
2
upon stimulation with P. acnes, counteract the growth of the acne.
bacteria. Those results highlight a new mechanisms by which
keratinocytes participate in the innate immune response to Materials and Methods
pathogens.
Finally, the inhibition of O N2 production, IL-8 release and Bacterial culture
2
keratinocyteapoptosisbyretinoicacidderivates,themostefficient P. acnes strain 6919 was obtained from the American Type
anti-acneicdrugs,demonstratestherelevanceofthesepathwaysin Culture Collection (Manassas, VA) and grown under anaerobic
vivo.Inaddition,ourdataareinagreementwiththeobservations conditionsinreinforcedclostridialliquidandsolidmedium(RCM)
that , retinoic acid can induce MnSOD mRNA in a human (DifcoLaboratories,Detroit,MI)at37uCduring5daysinorderto
PLoSPathogens | www.plospathogens.org 10 July2009 | Volume 5 | Issue 7 | e1000527