[10]J.Li,C.Sutter,D.S.Parker,T.Blauwkamp,M.Fang,K.M.Cadigan,CBP/p300are bimodalregulatorsofWntsignaling,EMBOJ.26(2007)2284–2294.
DaikiRokunohe,HajimeNakano*,EijiroAkasaka,YukaToyomaki,
DaisukeSawamura
DepartmentofDermatology,HirosakiUniversityGraduateSchool
ofMedicine,5Zaifu-cho,Hirosaki036-8562,Japan
*Correspondingauthor.
E-mailaddress:hnakano@hirosaki-u.ac.jp(H.Nakano).
Received24March2016
http://dx.doi.org/10.1016/j.jdermsci.2016.06.005
Letter
to
the
Editor
Lowdosecytokinesreduceoxidativestressin
primarylesionalfibroblastsobtainedfrom
psoriaticpatients
Psoriasis vulgaris is an inflammatory systemic disease with
unclearpathogenesis characterized by theappearance of scaly
erythematousplaquesontheskin.Theseplaquesarethesitesof
keratinocytes hyper-proliferation and misdifferentiation and
inflammatoryinfiltrationbyTcellsskewedtowardTh1/Th17with
respect to Th2/TREG. Such disequilibrium between T cells
population features psoriasis as an autoimmune disease [1]. A
prominentattentionisgiventobiologicdrugsaimedtorestorethe
balance between T cells populations in psoriasis: the use of
antibodies against the overproduced cytokines or the use of
cytokines-antagonists represent two successful strategies that
have been shown to ameliorate Psoriasis Area Severity Index
Fig.1.TotalROSproductioninintactprimaryfibroblastsobtainedfromthelesionalskinofpsoriaticpatients(LES)andfromhealthydonors(CTR).
TotalROSproductionmeasuredbyFACSAnalysisusingH2DCFDAfluorescentprobe(A,B)following48hincubationwith10fg/mlofsequential-kinetic-activation(SKA)IL-4
(LES+IL-4),IL-10(LES+IL-10),bFGF(LES+bFGF)orb-endorphin(LES+ bEnd).TreatmentofLESwithlowdosecytokinessignificantlysuppressedtheemissionofH2DCFDA
fluorescenceinthepresentexperimentalcondition.(C)QuantitativeanalysisofROSproductionbyflowcytometrywhereROSproductionisexpressedinpercentageversus CTR.Reportedvalues(meansSD)arerepresentativeoffiveindependentexperiments,eachoneperformedintriplicate.*Significantdifference(P0.05)versusuntreated CTR,xSignificantdifference(P0.05)versusuntreatedLES.
(PASI).Althoughtargetingspecificpartsofimmunesystem,such
treatments require the use of high dose biologics and are
frequentlyassociatedwithadverseeffects[2].
Thereareevidencesthatlowdosesofcytokinespreparedby
sequential-kinetic-activation (SKA) are effective and reduce
adverse effects in psoriasis treatment withrespect tostandard
doseofrecombinantcytokines.Thus,inaclinicaltrialinvolving48
patientsaffectedbypsoriasisvulgaris,theoraladministrationof
SKAlow-doseIL-4,IL-10andIL-11ledtoasignificantreductionin
PASI index with no adverse events [3]. Previously, signs of
oxidativestress were found in the blood and skinof psoriatic
patients[4,5]andaccordingtoourdata,anti-TNF
a
drugInfliximabreducesoxidative stress in Peripheral Blood Mononuclear cells
(PBMC)frompsoriaticpatients[4].Moreover,werecentlyshowed
thatthetreatmentwithlowdoseSKA(femtogramsperml)IL-4,
IL-10, basic fibroblasts growth factor (bFGF) and
b
-endorphin(
b
-End)decreasesoxidative stressin perilesional keratinocytesobtainedfromvitiligoskin[6].
InthepresentstudytheeffectoflowdoseSKAcytokinesin
fibroblastsfromlesionalskinofpsoriaticpatientswasinvestigated.
Wefocusedourattentionondermalfibroblastsbecausethesecells
providea crucial microenvironment for epidermal keratinocyte
function and, together with infiltrating polymorphonuclear
leukocytes wereshown to extensively produce superoxide and
H2O2 impairing the redox balance in psoriatic derma and
participating,atleastpartially,inabnormalkeratinocytesgrowth
inpsoriasis[7].
ThefollowingSKAcytokines(10fg/ml)wereused:(a)IL-10and
IL-4producedbyTREGsandTh2,respectively;(b)
b
-endorphin,neuropeptide that was shown tobe increasedin the serum of
psoriaticpatientsand produceddirectlyin psoriaticplaques by
inflammatorycells[8];and(c)bFGFthatwasshowntoincrease
cellular resistance to oxidativestress invitro [9].Lesional skin
punch biopsies were obtained from four patients affected by
plaque psoriasis and primary fibroblasts cell cultures were
established. Cells on passages 2–5 were used for experiments.
Cellularredoxprofilewasdeterminedbymeasuringintracellular
ROS production by flow cytometry analysis (fluorescent probe
H2DCFDA)andNADPHoxidaseactivitybyluminometricassay[4].
In Fig.1 asignificantlyhigher totalROS productionby lesional
fibroblastswithrespecttocontrolfibroblastsisshown17011%
versus 1007%) respectively. In lesional fibroblasts NADPH
oxidaseactivitysignificantlyraised(by51.4315%)comparedto
controlcells(Fig.2).
InordertoinvestigatetheroleoflowdoseSKAIL-4,IL-10,bFGF
and
b
-End on the above parameters, lesional fibroblasts wereincubatedwitheachcytokinefor48handthentotalROSproduction
and NADPHoxidaseactivitywere measured.Interestingly,every
cytokinetreatmentsignificantlyreducedintracellularROS
produc-tion(Fig.1):IL-4by29.33,IL-10by24.32,bFGFby24.34and
Fig.2.NADPHoxidaseactivityinintactprimaryfibroblastsobtainedfromthelesionalskin(LES)ofpsoriaticpatientsandfromhealthydonors(CTR).
NADPHoxidaseactivitywasmeasuredbyaluminometricassayfollowing48hincubationwith10fg/mlofsequential-kinetic-activation(SKA)IL-4(LES+IL-4),IL-10(LES+ IL-10),bFGF(LES+bFGF)orb-endorphin(LES+bEnd).AmongallcytokinesonlybFGFledtoasignificantdown-regulationofNADPHoxidaseactivityinLESfibroblasts.The histogram(C)representsthecorrespondingvaluesin%normalizedtoCTRofareaunderthecurveofNADPHoxidaseactivity.ExtracellularROSproductionwassignificantly higherinLESwithrespecttoCTRfibroblasts.Reportedvalues(meansSD)arerepresentativeoffiveindependentexperiments,eachoneperformedintriplicate.*Significant difference(P0.05)versusuntreatedCTR,xSignificantdifference(P0.05)versusuntreatedLES.
b
-Endby20.55%vsLESfibroblasts.AsregardsasNADPHoxidaseactivity, only bFGF among selected cytokines was effective in
reducingthisparameterinlesionalcellstocontrollevel(Fig.2).
Firstly,theresultsofourstudydemonstrateanincreasedROS
productioninlesional fibroblastsfrompsoriaticpatients.
More-over,weshowthatalsoextracellularROSproductionviaNADPH
oxidaseissignificantlyincreasedinlesionalwithrespecttocontrol
fibroblasts.Importantly,allexperimentswereperformedinvitro,
intheabsenceofTcellinfiltrates,soourdataindicatetheexistence
ofanintrinsicoxidativestressconditioninpsoriaticfibroblasts.
Inparticular,inpsoriaticlesion,extracellularROS
overproduc-tionbyfibroblastscanexertapro-inflammatoryroleinpsoriatic
skinindependentlyandinconcomitancewithinfiltratingTcells.
Hence,redoxbalancingagentscanrepresentanadjuvanttherapy
forpsoriasis.Inthecurrentworkwestudiedtheeffectoflowdose
SKAIL-4,IL-10,bFGFand
b
-Endontheredoxbalanceoflesionalfibroblasts. We found that all of selected cytokines decreased
significantlyintracellularROSproduction in lesionalfibroblasts,
however, only bFGF was effective in reducing NADPH oxidase
activity. The inhibitory effect of bFGF on NADPH oxidase was
already shown in adipocytesvia the mechanism of direct non
kinase-dependentcouplingofbFGFreceptortoGproteinofNADPH
oxidase[10].Asimilarmechanismcouldoccurinpsoriaticlesional
fibroblastsalthoughfurtherstudiesareneeded.Atthesametime
IL-4,IL-10and
b
-EndactonintracellularROSproducingsystems,such as mitochondrial electron transport chain or xanthine
oxidase:othertwomainsourcesofROSinthecell.Takentogether,
ourpreliminarydatashowtheeffectivenessoflowdosesSKAIL-4,
IL-10,bFGFand
b
-Endinmodulationofoxidativestressinlesionalfibroblastsobtainedfromtheskinofpsoriaticpatients.Although
furtherstudiesshouldbeperformed,thepossibleuseoflowdose
cytokinesasa targetingtreatmentfor psoriasisvulgaris canbe
suggested.
Conflictofinterest
Theauthorshavenoconflictofinteresttodeclare.
Fundingsource
Ente Cassa di Risparmio di Pistoia e Pescia (Grant no.
2011.0365). References
[1]A.Alikhan,L.M.Felsten,M.Daly,V.Petronic-Rosic,Vitiligo:acomprehensive overview Part I. Introduction, epidemiology, quality of life, diagnosis,
differentialdiagnosis,associations,histopathology,etiology,andwork-up,J. Am.Acad.Dermatol.65(2011)473–491.
[2]D.E.Branişteanu,C.M.Voicu,A.CreÛu,A.Dimitriu,M.C.Luca,C.M.Salavastru, Adversereactionsofbiologicaltherapyforpsoriasis,Rev.Med.Chir.Soc.Med. Nat.Iasi.119(2015)38–44.
[3]M.L.Roberti,L.Ricottini,A.Capponi,E.Sclauzero,P.Vicenti,E.Fiorentini,etal., Immunomodulatingtreatmentwithlowdoseinterleukin-4,interleukin-10 andinterleukin-11inpsoriasisvulgaris,J.Biol.Regul.Homeost.Agents28 (2014)133–139.
[4]V.Barygina,M.Becatti,G.Soldi,F.Prignano,T.Lotti,P.Nassi,D.Wright,N. Taddei,C.Fiorillo,Alteredredoxstatusinthebloodofpsoriaticpatients: involvementofNADPHoxidaseandroleofanti-TNF-atherapy,RedoxRep.18 (2013)100–106.
[5]Q.Zhou,U.Mrowietz,M.Rostami-Yazdi,Oxidativestressinthepathogenesisof psoriasis,FreeRadic.Biol.Med.47(2009)891–905.
[6]V.Barygina,M.Becatti,T.Lotti,S.Moretti,N.Taddei,C.Fiorillo,Treatmentwith low-dose cytokines reduces oxidative-mediated injury in perilesional keratinocytesfromvitiligoskin,J.Dermatol.Sci.79(2015)163–170. [7]K.J.Trouba,H.K.Hamadeh,R.P.Amin,D.R.Germolec,Oxidativestressandits
roleinskindisease,Antioxid.RedoxSignal.4(2002)665–673.
[8]W. Glinski, H. Brodecka, M. Glinska-Ferenz, D. Kowalski, Increased concentration of beta-endorphin in sera of patients with psoriasis and otherinflammatorydermatoses,Br.J.Dermatol.131(1994)260–264. [9]A. Burdzinska, U. Bartoszuk-Bruzzone, M.M. Godlewski, A. Orzechowski,
Sodiumascorbateandbasicfibroblastgrowthfactorprotectmuscle-derived cellsfromH2O2-inducedoxidativestress,Comp.Med.56(2006)493–501. [10]H.I.Krieger-Brauer,P.Medda,H.Kather,Basicfibroblastgrowthfactorutilizes
both types of component subunits of Gs for dual signaling in human adipocytes.Stimulation ofadenylylcyclaseviaGalph(s)andinhibitionof NADPHoxidasebyGbetagamma(s),J.Biol.Chem.275(2000)35920–35925.
VictoriaBarygina1,*,MatteoBecatti1
Department of Experimental and Clinical Biomedical Sciences
“MarioSerio”,UniversityofFlorence,VialeMorgagni,50,50134
Florence,Italy
TorelloLotti
Dermatology and Venereology Division, Guglielmo Marconi
University,ViaPlinio,44,00193Roma,Italy
NiccolòTaddei,ClaudiaFiorillo
Department of Experimental and Clinical Biomedical Sciences
“MarioSerio”,UniversityofFlorence,VialeMorgagni,50,50134
Florence,Italy
1
Theseauthorscontributedequallytothiswork.
*Correspondingauthor.
E-mailaddress:v.barygina@gmail.com(V.Barygina).
Received1April2016
http://dx.doi.org/10.1016/j.jdermsci.2016.06.002
Letter
to
the
Editor
Associationofinterleukin(IL)18andIL10gene
polymorphismswithorallichenplanusrisk;a
case-controlstudy
Expressionofbothpro-andanti-inflammatorycytokineshas
beenreportedinorallichenplanus(OLP)[1].Interleukin-18(IL18)
aninterferongamma(INF
g
)inducingagentisinvolvedinOLPasitisapotentpro-inflammatorycytokinebesidesitsinhibitoryeffect
onthesynthesisofIL10[2].IL-10isananti-inflammatorycytokine
having a direct ability to down-regulate IL18 and INF
g
, whichmakesitanimportantimmunoregulatoraswellasamediatorof
inflammatoryprocess[3].
Genetic factors producing disproportions in pro- and
anti-inflammatory cytokine production have been anticipated to
increasethesusceptibilityforOLP[1].
Theabovementionedentangledcytokinesnetworkconnections
drewourattentiontoinvestigateapossibleassociationbetween
functionalsinglenucleotidepolymorphisms(SNPs)relatedtothese
cytokinesandOLP.Theimpact ofthesepolymorphismsontissueIL18
andIL10expressionwasalsodetermined.
Thiscase-controlstudyincluded72consecutive newly
diag-nosedOLPEgyptianpatientsand75apparentlyhealthyEgyptian
volunteersascontrols. Allsubjectswerefreefromany systemic
