Concentration of Filaggrin Monomers, Its Metabolites and Corneocyte Surface Texture in Individuals with a History of Atopic Dermatitis and Controls

University of Southern Denmark

Concentration of filaggrin monomers, its metabolites and corneocyte surface texture in individuals with a history of atopic dermatitis and controls

Engebretsen, Kristiane A; Bandier, Josefine; Kezic, Sanja; Riethmüller, Christoph; Heegaard, Niels H H; Carlsen, Berit C; Linneberg, Allan; Johansen, Jeanne D; Thyssen, Jacob P

Published in: Journal of the European Academy of Dermatology and Venereology

DOI: 10.1111/jdv.14801

Publication date: 2018

Document version: Accepted manuscript

Citation for pulished version (APA): Engebretsen, K. A., Bandier, J., Kezic, S., Riethmüller, C., Heegaard, N. H. H., Carlsen, B. C., Linneberg, A., Johansen, J. D., & Thyssen, J. P. (2018). Concentration of filaggrin monomers, its metabolites and corneocyte surface texture in individuals with a history of atopic dermatitis and controls. Journal of the European Academy of Dermatology and Venereology, 32(5), 796-804. https://doi.org/10.1111/jdv.14801

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Download date: 27. Sep. 2021 10 9 8 7 Denmark Denmark, Odense, 6 5 4 Netherlands Amsterdam, DEThe 1100 of Amsterdam, University institute, Healthresearch Public Amsterdam Center, 3 Denmark. Hellerup, 2 Denmark Hellerup, of Copenhagen, University 1 passed † ¶ Linneberg Riethmüller Kristiane A individuals with ahistory Concentration of Article :Original Article type This article isThis article by protected copyright. All rightsreserved. 10.11 differences thisbetween andVersion version the ofRecord.cite Please this asdoi: article the through copyediting, typesetting, pagination and proofreading process, to whichmay lead This article acceptedhas been interests of Conflicts of themanuscript. preparation Foundation. Lundbeck the from grant StanDerm. the from and sources Funding head: Running

Department Research Denmark Copenhagen, Hospital, University Bispebjerg Dermatology, of Department Southern of Hospital,University University Odense &Pharmacology, Biochemistry Clinical of Department of Department Serend Health, Occupational Institute of Coronel Copenhagen, of University Hospital, Gentofte Herlev and Allergy, and Dermatology of Department National

Equal responsibilities asauthors first responsibilities Equal Department This work is dedicated to our dear friend and colleague, Niels H. H. Heegaard, who suddenly and suddenly who H. Heegaard, NielsH. colleague, and friend dear toour isdedicated work This

11/jdv.14801 Accepted Article a - ip GmbH, Centre for Nanotechnology, Münster, Heidelberg, Germany Heidelberg, Münster, Nanotechnology, for GmbH,ip Centre way. Allergy Research Centre, Department of Dermatology and Allergy, Herlev and Gentofte Gentofte and Herlev andAllergy, Dermatology Departmentof Centre, Research Allergy 8, Centre for Prevention and Health, Centre for Health, The Capital Region, Denmark. Region, Capital The Health, for andHealth, Centre Prevention for Centre 9 . Engebretsen . Jacob P. Thyssen and Kristiane Aasen Engebretsen are financially supported by an unrestricted unrestricted an by supported financially are Engebretsen Aasen Kristiane and Thyssen P. Jacob 4 . ,10

of Clinical Experimental Research, Rigshospitalet, Glostrup, Denmark. Glostrup, Rigshospitalet, Research, Experimental Clinical of

PhD, PhD, of Clinical Medicine, University of Copenhagen, Copenhagen, Denmark. Copenhagen, Copenhagen, of University Medicine, Clinical of Aage Bang Foundation. Bang Aage

MD, PhD, MD, Filaggrin and Filaggrin Autoimm : The project has received financial funding from funding financial received has project The Niels

filaggrin

: The auth The H.H. He H.H. ¶

unology & unology Jeanne D. 1 ,2

MD and JosefineMD and Bandier corneocyte corneocyte surface

of atopicdermatitis

ors have no conflict of interest to declare. to of interest conflict have ors no monomer

egaard for publication andundergone fullpeer review buthasnotbeen

Johansen Biomarkers

Furthermore † 5 The funding sources did not play any role in the study or the the or study the in role any play not did sources funding The ,6 s Amsterdam Health Public

, MD

its 1,2

MD, DMSc metabolites DMSc , Statens S , Statens texture

the study was study the

and controls ¶ , DNatSc 1

,2 in in

MD, dermatitis erum Institut, Copenhagen, Denmark Copenhagen, erum Institut, , Jacob P. , Jacob

and

PhD, ,

Berit C. Carlsen C. Berit

Copenhagen County Research Foundation Foundation Research County Copenhagen

corneocyte surface corneocyte surface texture

s Sanja KezicSanja

upported upported Research Institute,

Thyssen

1,2 by the COST Action TD1206 TD1206 Action COST the by

3 MD,

PhD 7

MD, PhD, PhD, MD,

, PhD, PhD,

Christoph Academic Medical Academic Medical DMSc

Allan

in Hospital, Hospital,

sadly sadly patient population,a Methods: patients with controlled Objective: far examined association the between parameters these and in filaggrin gene ( Dermal Index (DTI) Texture number of Background: Abstract E 38 677101 +45 Fax: 31 50. +45 3867 Phone: DK Kildegaa Herlev Dermato of Department DMSc PhD, MD, Thyssen, Jacob P. Correspondence: publication. presentations Prior This article isThis article by protected copyright. All rightsreserved. significantly inthe higher oldest age group compar palmar xerosis, phenotypes such as hyperlinearity andAD. Among healthy controls, DTI was of monomeric filaggrin mutation Results: Transepidermal loss water Criteria - mail: [email protected] monomeric filaggrin - Accepted Article Denmark Hellerup, 2900 and Gentofte Hospital Gentofte and . rdsvej 28, 28, rdsvej

s All forparticipants were tested In the patient population,In thepatient 78.7%hadahistory (37/47) had ofAD 59.5%(28/47) and

. A total of67

nano P To atients had significantlyatients had ofhigher levels levels DTIandsignificantly lower of

Atopic dermatitisAtopic (AD) characterized byskin is dysfunction. barrier Notably, determine -

scale

: The content has not been published previously and is not otherwise submitted for for otherwisesubmitted isnot and previously published been has not content : The FLG

personal history of and NMF compared to and to NMFcompared the 20 healthy controls. logy and and logy protrusions on surface the of corneocytes )

, and adults adults

dermatitis. and reduced

DTI ,

monomeric filaggrin NMF correlate , , was recently Allergy, (20 healthy controls and47 monomeric filaggrin

levels oflevels moisturizing natural factors (NMF)

FLG ADwas

d associated

with thepresence of

mutations (R501X, 2282del4 ,

diagnosed by U.K.diagnosed the Diagnostic Working Party’s DTI and ed to ed to twothe youngeragegroups.

and

with NMF

NMF derm pediatric monomeric filaggrin

in healthy controls and , which atitis atitis

were measured FLG AD Among patients

mutations and mutations can be ,

loss ,

R2447X). patients, reduced patients, reduced level ) -

of expressed expressed were included . -

levels inadults. function mutations .

No study has No studyhas so

clinical a group a group of by

the a high .

In the FLG

higher higher in bothnon Dermal R identified projections’ may appear impairmentbarrier corneocytes matrixin a of Human Introduction with age. individuals in healthy the c toinflammation isknown filaggrin reduce levels. dermatitis or non comparing dermatitis Conclusion: This article isThis article by protected copyright. All rightsreserved. biomarkers impairment, need there is a for AD Since (NMF),factors inverse ofpresence common e Accepted cently, Article

correlation stratum (SC)consists corneum of Texture Index Index (DTI) Texture

. , a methodnovel

and other formsand other dermatitis of

between between (Figure 1)(Figure or ‘circular nano or ‘circular

was significant difference inDTI, - visible visible significantinflammation hasa andnegative effect onthe asskin barrier - lesional from andlesional skin between DTIandthedegradation p and

observed dermatitis loss patients with controls healthy . 2, 2, 3

atopic dermatitisatopic (AD), -

These protrusions of was developed hydrophobic hydrophobic ontrol group,suggestinga , function mutations function in filaggrin gene ( - . 4 was established objects’,

additional

patients controls and

but lipids, resembling andmortar’ ‘bricks ,

is characterized by skinprimary orsecondary barrier to studies examine differencethat the various in multiple layersofprotein neither accurately count have monomeric filaggrin to enable nano in the pastin the DTI was significantly increasedin DTI wassignificantly aged pediatric pediatric the .

. - These findings suggest that even suggestthatThese findings even mild

scale scale For example, cytokinesthat are active in ir gradual roducts offilaggrin

function compar

protrusions AD an patients been described as‘villus these these change change in corneocytemorphology ative studies ,

n FLG

or and NMF corneocyte protrusions, the - enriched, the o ) . n ir 3,

the surface corneocyte , natural moisturizing, natural

4 composition d correlated with

Furthermore

levels . . flattened 1 Importantly,

In case ofskin

was found - like

and smooth has been has been novel novel ,

a strong DTI

the when when

was carriers ofcarriers common monomeric filaggrin etBandier al. and GentofteHospital, Denmark. medical ofpatientswithdermatitis charts attheDepartment ofDermatology Allergy and co with controlled disease A Study population andMaterial methods monomeric filaggrin monomer levels. innate and This article isThis article by protected copyright. All rightsreserved. patient asforthe controls population. current cortico psoriasis) eczema rheumatoid (i.e. widespread

ntrols by wererecruited advertisement online Accepted Article of67total steroids or ,

past or acquired immune of response AD down dermatitis Caucasian Caucasian

underwent 6

history of within within That 5

In the In the

FLG

study demonstrated adose levels and and and NMFinhealthy controls

2 weeks (mild eczemawasaccepted) )

individuals participated studyparticipated in the current arthritis, Crohn’sarthritis, disease,ulcerative colitis,systemic erythematous lupus or ultraviolet ultraviolet

dermatitis mutations. ,

or used FLG study, we Participants were invited , otherwise the applied forthehealthy same criteria exclusion (UV) aged 18 6

mutations and included both homozygous andheterozygous

Patients Blood samples were Blood sampleswere fromcollected for healthycontrols the systemic irradiation 3weeks irradiation before determined - 68

were

- . years dependent correlation between epidermal

The patient population was recruited by immun between Octoberbetween 2011and 2012 March - , other , other chronic inflammatory besides diseases in regulate regulate eligible for participation eligible for participation ifthey hadactive or (20 healthy controls and 47 DTI as well asthe well of quantity DTI as epidermal dermatitis o

suppressant in connection with FLG

patients expression and reduce and filaggrinexpression

study start s . The

with controlled disease healthy

a ,

had previous study by

dermatitis

controls had no used topical . Healthy . Healthy , reviewing Herlev

patients .

TEWLwas measured Transepidermal of tothe AD according U.K.Work if they keratosis pilaris J Clinical population fortested three Diagnostic Criteria patientIn the population, a dermatitisAtopic andfilaggrin skin measurements and sampling. FLG This article isThis article by protected copyright. All rightsreserved. calculated (g/m punch where the biopsy for epidermal electronics GmbH, CKelectronics, UK) . B andier 2 genotyping Accepted)/ Article h) ever assessment

was measured30s for assessed

s and the usedin statistical analyses

had experienced (R501X, 2282del4and

of the . Furthermore, water loss .

The

and registered and represented ‘AD ever’

of the participantshistory and atopic of disease

most common with the MPA5with the Probesystem, (Multi Adaptor Khazaka and Courage participants instructed were not

personal historypersonal of

fissures hands ontheir and

participants were participants asked they if had ahistory of asthma genotype econds clinical

ing Party’s Diagnostic Criteria.ing Party’s R

FLG 2447X) by multiplex 2447X) by filaggrin

and the average of the 10 last seconds average ofthe10last and the ofsteadystatewas

skin loss

the the . -

. features inner surface ofthe AD of 7

monomer Current ADwas assessed. Current not - function

was diagnosedby to

such as

feet use moisturizers

quantification was taken). wasquantification taken).

mutations PCR

a nd questions related to thediagnosi xerosis, analysis 7 left left

among the U.K.the Working Party’s and symptoms upper arm palmar and hyperlinearity . 8, 8, 9 at the day the at of

Northern European All (opposite side (opposite of participants were

TEWL

hay fever

s ,

finalgave the DTI. images morphometrically andtothem filter and by size shape Germany).GmbH, Munster, was analyzedbyusi (0.01were used N/m, MLCT,Mannh VEECO, Instruments, Barbara,CA, Santa USA) controllerMultimode withthesoftwareversion5.30sr3(Digital NanoscopeIII AFMequipped and was analyzedby until analysis Inst forearm. appliedfor5sbytheuseof were apressure applicator (D500 Tapes Squame, CuDerm,Dallas, TX,USA) wereattachedhealthy to ornon wereCorneocytes sampled by morphologyCorneocyte andsurface DTI This article isThis article by protected copyright. All rightsreserved. ModelVibrax 2200,IKA by tape acid (PCA)andcarboxylic urocanic (UCA)acid (trans fourth the determine were bythesameCorneocytes collected technique described stripping as tape previously to Determination offilaggrin degradation products

Accepted Articlerum of 20 of 20 ent, ent, TX,USA),CuDerm, Dallas adding

consecutive tape. the amountdegradationfilaggrin products, ofand the analyses were . µ

The sameskin location wastape m 500 µL25%(w/w)ammoniaAfter 2hours solution. of

2 Atomic Microscopy Force (

were assessed, andwere assessed, the

ng the nAnostic method with

- works Inc, Wilmington,works Inc, extracts NC, the were evaporated USA), to 13 12

Briefly,

a Computer visionwas to used eachevaluate nanostructure tape tape stripping techniquestripping where adhesive round, (3.8tapes cm the the

and 10

mean ofthe value identified objects inthese 10images

NMF components histidine (His), 2 gently removed b s ilicon AFM - eim, T Germany). stripped five consecutive times, th and

in stratumcorneum custom ) -

nitride nitride tips onV as

. described detailselsewhere. in -

For eachsample, 10randomly selected

and cis isomer) w -

built proprietary algorithms (Serend y tweezers y he to - shaped gold

- pographic cell surface data lesional on skin volar the , continuous continuous

and vial stored in aclosed ere

– extracted - pyrrolidone

also also D - - coated cantilevers Squame Pressure Squame Pressure shaking (IKA performed on 11 e fourthtape

from each Br iefly, - 5 - 2 - , D ip

a - -

immunosorbent assay immunosorbent assay (ELISA) Quantification of the use of peeled potassium 2mM phosphate, Na of upper the arm. Epidermal m Quantification ofepidermal mmol/gwere expressedas kit(Thermoassay Scientific, Fischer Rockford, USA) IL, KOH inbothwas performed. measured extractions were bya Proteins determined. amountvariable ofstratum proteinonthetapestrips, corneum total the amount was of protein residu at60°Cdryness (EppendorfConcentrator5301, This article isThis article by protected copyright. All rightsreserved. The independent sample t patientIn the population, of and levels To determine whether the were data normally distributed Statistics expressed as AU/mg epidermis.

Acceptedmincing,of Article e was dissolved e was dissolved in 500

after after incubation

Due toincompleteextraction ofproteinsbyammonia, asecond e

onomeric filaggrin monomeric filaggrin extraction buffer 6

B iopsies monomeric - NMF andDTI test wasused compare to groups, two while at total

were monomeric monomeric filaggrin

µL Millipore water µL Millipore and analyzed byHPLC. 56°C for 10min 2

as previously described as previously

EDTA, pH 7) and placed at EDTA, pH7)andplaced protein was quantified

, grinding, stored Eppendorf in vials filaggrin level was performed by

and NMF . 13

data weredata

sonication are

and then in

Eppendorf AG, Hamburg, Germany)Eppendorf AG, Hamburg, presented asmedianpresented values

in the skin in the 4 mm

log transformed normal obtain distribution. to ,

. centrifu the the e 6 skin

, The of level

we Shapiro used the added together,andt added containing -

pidermal protein wasextracted bythe 80°C until 80°C until analysis. biop gation s

ies ies taken from the an

, delipidation anddialysis

in monomeric filaggrin storage buffer (10

o Pierce Micro BCA protein - ne house enzyme

To compensate for the To compensateforthe - way

(25th/75th percentiles) xtraction with0.1M - Wilk test he levels ofNMF ANOVA E pidermis was

inner surfaceinner and the and the - linked linked

. mM

The followed followed

was DTI .

NY, andGraph USA) performed. designexplorative and experimental of study, the nocorrection ofmultiple thesewithout features. DTIwould haveahigher healthy controls. Furthermore patients weexpected that with higher variablefrom reference the in linearthe regression. NMF wereasdependent used variables by The influenceof confounders possible independentthe samples t onDTI,monomericdata filaggrin and NMFweretransformed log forcomparisonswere used oftwo andmultiple groups, groups respectively. Mann by This article isThis article by protected copyright. All rightsreserved. ofAD and59.6 history andpopulation healthy the (P=0.553). controls (range29 years 20 18 (range T Results he patient population

using using Tu - Accepted Article hoctestwaskey post for used multiple comparisons. , Whitney and Utest

and the of level

multiple linear regression model - 67) All statistical analyseswere .

T he control group he control - 68 years). P % (28/47) had% (28/47) least one at ad Prism Software, 7(GraphPad Inc, SanDiego,Calif). P consisted of consisted monomeric filaggrin - , values <0.05values be to wereconsidered statisticallysignificant.

and lower and lower oflevels Kruskal test or the one No statistical difference patient the in age was found between had -

Wallis test Wallis 18males and 29 females

10 male10 and10female participants ,

. respectively,

performed withIBM 22 SPSS Statistics (IBM, Armonk, The exponentiated coefficient - s way ANOVA followed byTukeyposthoc test wasused. ,

where th

and NMFlower

monomeric filaggrin with pairwise Mann In the patient In thepatient population, 78.7% (37 FLG

Our e age, sex, age, sex, AD and

log

mutation a priori hypothesisa priori wasthat

- transformed ofDTI, f data For , ,

FLG

and the medianand the was age 41 years in dermatitis patientscompared to monomeric filaggrinmonomeric

( 20

mutations withAD history anda -

to obtain normal distrib to obtain and NM Whitney U

heterozygote F wa LG ,

s reported as%change s reported and the medianand the was age

comparisons F

mutations In the healthyIn the controls, ,

compared to patients compared to -

test hoc post s

and /

47) a had DTI would be ilaggrin and ilaggrin levels Due tothe ,

was assessed 8

was ution , the the

tests

and

found. younge When for andoldest adjusting age grouphad74% sex,the DTI higher comparedvalues to the (<30age group years) group, ( Overall, the patient populationhadsignificantlyhigher DT (DTI)Dermal TextureIndex controls feet. controlgroupthe ofasthmhistory 44.7% (21/47)had xerosis,clinical 66.0% (31/47) hadkeratosis63.8% pilaris, h This article isThis article by protected copyright. All rightsreserved. P=0.001) controlgroupthe ( Overall, t E sex. patient population median omozygote pidermal pidermal

Accepted ArticleThe median values of NMF and DTI,filaggrin

No significant differencesNo significant inDTI population werefoundforother characteristics the inthe DTI st age(P<0.0001). group

according to studyaccording to population are characteristics listed inTable DTI

he patient populationhadsignificantlylower levels of (Figure monomeric filaggrin

was s ; )

. 25 Furthermore a or hayfever,and a or significantly highersignificantly th had clinicalxerosis, palmar orahistoryof hyperlinearity onhandsand fissures 2 /75

AU/mg epidermis; 25 or the groupcontrol ) a history onhandsandfeet. offissures . In the patient population,In thepatient p th

(P<0.0001) and between30 those percentile, , 61.7% (29/47) had a history ofasthma hada, 61.7%(29/47) history (14/47) had hayfever,29.8% or

In the patient population,In thepatient no associationDTI agewas between and

75; vs.4 59/98 25

in .0

the oldestthe agegroup(>50year in % (5/20) had keratosis pilaris. None% (5/20)hadkeratosispilaris. oftheparticipantsin th

/75 the unadjusted afterthe analyses or adjustment th

percentile, 58.7;16.5/180.5 108.1/486.3,vs. 195.2; articipants 4 ;

31/78, in the patient and in the population in healthy the - 50 years ofage(P<0.0001)

In the control groupIn thecontrol 15

P=0.015) I valuescompared to control groupthe with (30/47) hadpalmar and hyperlinearity monomeric filaggrin FLG

(Figure

s) muta

. compared the to youngest

tions had 2 ) . I n the control .0 % (3/20) hada % (3/20)

significantly

(

compared to for ageand Figure 3 Figure ) .

history of history 1)(Figure groupcontrol ( Overall, the of weresignificantlylevels patient NMF lowerthe compared in population to the degradationFilaggrin products orpopulation the group. in control significant differences inthelevelsof foundwith filaggrinregardstosex were ageor (P forage adjusted showedAnalyses andsex that compared without those such to fil wild types and Wh shown level lower This article isThis article by protected copyright. All rightsreserved. in the (P=0.0 sex compared heterozygous to (P=0.004) wildtype and (P=0.0 cases forstratified number the ofmutations, homozygous had carriers significantly lower NMF levels unadjusted FLG = aggrin , homozygous carriers hada en s

0.001) the homozygousmutationthe carriershad9

Acceptedmutations, ahistory ofADwas associated with a Article unadjusted 1 by Bandier etal. by Bandier tratified forthenumbertratified of 0

levels were significantlylevels were lower ). .

and in the s In thepatient A history ofasthma withA history hayfeverwasassociated significantly or lower ofNMF levels analyses, no forthedifference wasseen presence of any

compared to palmar hyperlinearity mmol/g 25 protein; monomeric filaggrin age and sex analyses (P=0.020)after and adjustment for andsex age reduction, (17% ,

6 population, population, t

and this was

participants without AD (P adjusted analyses

24 (NMF) clinical

mutations, heterozy th

% reductioninNMF levels compared wildtype to /75

in with a with a he NMFlevel th the the also evidentalso

characteristics in percentile, 0.66; 0.47/0.96percentile, 0.66; v

unadjusted participants withparticipants 59 0 % lower levels of % reduction(P

xerosis (P = 0.00

s after adjustmentafter for andsex age

gous n

analyses =0.027), and when adjusted for andwhenadjusted sex =0.027), age, and

was associated ere in the

9 18% inNMFreduction (P=0.045)

and P<0.0001,respectively)

significant mutation carriersmutation had4 xerosis = unadjusted unadjusted 0.00

(P<0.0001) monomeric filaggrin 20). When 2

FLG and ) s ly

. 1.01; of filaggrin levels. with a 6

lower in participants withlower participants in palmar hyperlinearity

analyses mutation, butmutation,

(Table 1) adjusted foradjusted and age

0.78/1.17, P=0.005) 5 % reduction % reduction (Table 2 % lower levels% lower

(P< , as previously

compared to

cases . in patient the Monomeric w 0.0001). No hen 1 . )

. In the In the

a history of history ofpresence the significantly T findingsMain Discussion thoselowerof to with TEWL NMF compared values (g/m compare group (P=0.023). and inthegroup (<30 years) unadjusted analyses (P=0. were found sex (1 P=0.015). This article isThis article by protected copyright. All rightsreserved. who dermatitis. investigated relationshipthe changesin between The Interpretation compared twoyounger the agegroups. to he se FLG

increased DTI 47 2 developed developed

)/h), defined )/h), ashigherthan the median had ofallparticipants, value significantly levels lower patients,

Accepted Article6

% reduction, P=0.0 patients d to

mutations, NMF the agegroupoldest had28%higher levels compared the to youngest age AD Furthermore, was withxerosis associated lower ofNMF levels In . The oldest . Theoldest agehad (>50years) group NMF higher levels compared the to youngest age FLG

fe lower levelsoflower . 18

reduced reduced male (P=0.029) participants Among healthy

dermatitis with dermatitis under

mutations and In the group,In the control ealthy newborns observed levels of had a tendency ofhigher 25 monomeric filaggrin in patients with ) .

With control clinical phenotypes as such monomeric filaggrin followed from birth and followed from and birth upto regards regards to control s male parti , DTIwas significantlyhigherin oldestage the group

. Finally, participant dermatitis is

had sex, differences nosignificant inthe NM corneocyte cipants had cipants significantly lower

and NMFcomparedtothe significantly higher significantly higher levels ofDTI 017

number of

(P=0.033) and ), and

in withline previous NMF seemedto correlate xerosis, xerosis,

morphology when adjusted for sex, a history ofADwhen adjustedforsex,ahistory

‘villus s with highs with 12 TEWL (≥ values .

6 months palmar hyperlinearity - like projection

and when adjustedforageand ,

s 20 tudy subjects the presencethe of studies that

healthy controls levels of NMF

with and s ’ compared to

F levels F levels the the

and (n= 5 ) .

observe asignificant decreaseobserve in youngloricrin compared to s decreas proteins in involved the keratinization process alterations, but hypothesized al. Gorzelanny et cellsize increased described previously DTI age. between and healthy skin To ourknowledge ofactive dermatitisabsence andfurthermore theindividuals that population healthy controls (529lesional ±277 whodidthose notdevelop AD. This article isThis article by protected copyright. All rightsreserved. Accepted filaggrin to skin keep the hydrated. compared twoyounger the agegroups to (P=0.023), acompensatory perhaps dueto ofbreakdown patientpopulation,howeverthe which couldfor account differencethe with age. healthy skin. For example, measure wedidnot of amountordistribution lipidsaround cells the than factors monomeric deficiency filaggrin are responsible forthehigh DTI observedinage Article ed levelsoflipids anddown and . A

th small

(24 ±21 e healthycontrols as ‘humps’. , , we are the , weare first to describe

a rougher cellsurface counts .

study Thus 3

Nevertheless

counts ) ,

including 21healthy individuals aged1 and non a kin, supporting this assumption. that that ged corneocytes ged corneocytes 14

2 , The a

)

Furthermore, e we observed we observed . levels offilaggrin monomersother age. that with This suggests they were 3

In our study

- was - regulation ofregulation important epidermal such proteins as filaggrin and , lesional (116 ± 53 uthors morphological not as pronounced,not as ,

and

due to could notexplain nature the of nano these significantly of higherlevels NMF in compared young to corneocytes . Previous studies shown have has thataged skin possibly corneocytepossibly protrusions, describedby significantly , levated

the observedthe differencebetween No association betweenageandDTI wasfound in decreased production of and epidermal lipids

changes ofhave agedcorneocytes

levels of counts

increasing levelsofDTI age with which could beexplained bythe 15 ) - 17 pediatric DTI

were adults. In -

the current current the study 77 years, found

ha ve AD

been found in both been foundinboth skin

ha ve the the when when

significantly older patients older no , patient

- we did not we didnot scale scale

correlation been compared

in to The reduced levelsofThe reduced epidermal inflammation heterogeneity population ofthepatient mutations, detect (villi) to potentexposure the contact allergen 2,4,6 study in a SCORAD per se filaggrin NMF than withassociated AD affected this Howeve relationship. shown havebeenproposed. hypotheses of The nature thealtered topography of surface corneocyte still the is This article isThis article by protected copyright. All rightsreserved. of As expected, barrier negativelyaffect inflammation skin could and the in turn no ormild dermatitis which wasunder control downregulate IL such as healthy areinaccordanceto the controls withprevious research. Severalinflammatorymediators, common ,

Accepted Article. This

a significantdifference inDTI which disappearedtreatment after moisturizers with in children within children convalescent acuteand AD or NMF . - by a

4 the 4, IL xerosis or elevated TEWL

FLG Moreover, Moreover, the of level hydration skin notion notion 22, 23 22, .

filaggrin expression SCORAD

Matsumoto al. et -

13,

mutations. t could be could he levels ofNMF werelower in participants with was supported by factthat the IL - 17A,

(SCOring Atopic Dermatitis)(SCOring Atopic

more corneocyte foraltered important morphology than inflammation

IL - 18

22, monomeric filaggrin While a

, Here, 5, 19, 20 5, 19, IL

levels in the patient beduetothein the population. Thisfindingcould - , use of emollients or , useofemollients or 25, IL

experimentally

even in even in non strong

according to - trinitrochlorobenzene ledto corneocyte protrusions - , 33, TSLP

hereby thateven suggesting sub inverse the the might , 4

- it

DTI wasnotaffected byachange in lesional atopic lesional atopic skin. and NMF patient co inthe population r, t

cannot value correlation between DTIandNMF induced xerosis and TNF and be importantfor a history ofAD, he DTI

In thecurrentstudy, the the , suggesting absence that the of

be outruled skininflammation that presence of

reduce level - α have been shown to beenshown α have a history of

unresolved, but several

was relatedto more closely filaggrin and NMF and inflammation

DTI 21 the presencethe of

Our study had subjects sub

levels - - AD w clinical clinical e were

, as and

suggested

in carriers

un FLG by

was levels mpared able to

before sampling. we Finally, had noinformation emollient use, about exposurebathing within sun or weeks habits Signifi No information ADwas oncurrent collected been stored 2011, since but wedo established. anatomical variation. fromcollected inner on the ofsurface thearm, upper the while tape design filaggrin without with and To andStrengths limitations This article isThis article by protected copyright. All rightsreserved. suggesting properties.barrier dermatitis patients was found with between In this Conclusion our knowledge Acceptedmultiplecorrection for comparisons wasperformed Article . cant cant beresults should therefore interpreted withcautionmay asthey on bebased chance. human adult The for biopsy

levels ,

or a change steady incorneocyte morphology withage.Futurestudies need to identify the

It ispossibleIt thatabsoluteof levels DTI even subclinical inflammation

the volar aspect ofthe aspect volar forearmthe the to close and NMF

dermatitis , DTI was

this first studyis the toinvestigate stud

Also, analytical the variability oftheDTI measureyet hasnot been monomeric filaggrin y

, in the samein the group

a significant differencea significant inDTI, epidermal , significantly significantly increased agedindividuals in inthehealthy group, control

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not

expect the relative expect therelative levels tohavebeen affected.

and no clinical scoring ofeczema was performed severity

. , quantification and TEWL the takenmeasurements were

There aresomelimitations with regardsstudy tothe dermatitis and healthy controls is sufficient to to is sufficient

strips usedstrips todetermine DTIandNMFwere and NMFwere reduced since tape had strips DTI in a

despite ahighnumbertests. ofassociation despite

elbow produce alterations in produce important alterations skin

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monomeric filaggrin mutations ly to leading large large , indicating mild that , population of adults monomeric

bias

regarding and NMF .

tapes is influenced by pressure, time and anatomical location: integrity and cohesion assessed by by assessed cohesion integrity location: and timeandanatomical pressure, by tapes isinfluenced 10 2011; Med. J NEngl diseases. 9 1488 2012;413; Acta. Chimica Clinica genotyping. for applicable method auniversally offilaggrinpolymorphisms: multiplex analysis 8 dermatitis. atopic for ofdiscriminator set aminimum of I. Derivation dermatitis. foratopic criteria diagnostic 7 1299. 1296 2016;136; Dermatol. JInvest Irritation. to Skin itsResponse and Skin in Human Filaggrin Epidermal of 6 2014.27 BerlinHeidelberg, Springer Berlin, Heidelberg. Management. and Aspects Clinical Epidemiology, Science, Basic Filaggrin: editors. HI, Maibach Thyssen JP, 5 1573 2015;136; Immunol. JAllergy Clin dermatitis. withatopic patients in conformation corneocyte determine products breakdown 4 disease. skin indicate corneocytes 3 361 2013;19; ResTechnol. Skin barrier function. assess inf orwithout infantswith normal skin in the facial from corneocytes 2 328 2005;6; Biol. Rev MolCell 1 References betweeninterplay and cells matrix skin in ofDTInature This article isThis article by protected copyright. All rightsreserved. 477 2013;35; Sci. JCosmet pH.Int surface skin content and sebum hydration, SC TEWL, including 300people of 15 391. Exp corneum. nativestratum of fornanoanalysis tool aninnovative as microscopy 14 2144. ofnatural determination 13 TGF in force follows Nanotopography 12 895 2007;107; Ultramicroscopy. mapping. elasticity byAFM be identified 11 contro randomized, A stripping. tape sequential

483.

Accepted Article

Br J Dermatol. 1994;131; 383 1994;131; J Dermatol. Br

Meldgaard M, Szecsi PB, Carlsen BC, Thyssen JP, Johansen JD, Menné T, etal. Menné JD, JP, Johansen Thyssen BC, Carlsen PB, Szecsi M, Meldgaard T J,et al. Hunter M, Shipley R, ArcherC, P,Hay Williams H,Burney Bandier J, Ross De C, Vestergaard HaftekM,etal. J, R,Franz Abdayem KoppesSA, McAleer MA, Riethmuller C, of alterations Nanoscale S,etal. Kezic Thyssen JP, K, KramerA, Gevers J, BeutelM, Franz in H.villus Changes M,Mitsuyoshi H,Fukuyoshi O,Satoshi Naoko G. R,Melino Schmidt Candi E, Luebberding S, Krueger N, Kerscher M. Skin physiology in men and women: in vivo evaluation vivo women: in and inmen Skinphysiology Kerscher M. S, KruegerN, Luebberding Atomicforce SW. Schneider TA, K, Luger Thomas EM, T, Goerge Schnaeker C, Gorzelanny the for method HPLC an of A.Evaluation S,Kammeyer NL, Kezic I,Yau Dapic I,Jakasa J,O B,Wegener Reiss G, Thoelking can structures Vacuolar H. Oberleithner Stracke W, Kienberger F, TE, Schaffer Riethmuller C, adhesive by disruption Acute barrier JW. J, P,Fluhr M, Elsner Prassler Flach M, Breternitz allergic and with skin associated mutations Filaggrin DY. McLean WH,Leung Irvine AD, - rela ted corneocyte changes including moisturizing factors in the human stratum corneum. Anal Lett. 2013;46; 2133 2013;46; Lett. Anal corneum. stratum human inthe factors moisturizing - 1580 e1571 1580 365; 1315 365; - - - Han 1492. 340. leuran MS. Inflammatory leuran sen K, Carlsen BC, Tanassi JT, Johansen JD, Heegaard NH. Quantification Quantification NH. JD, Heegaard JT, Johansen Tanassi BC, Carlsen sen K, Skin Res Technol. 2016;22; 174 2016;22; Res Technol. Skin

- beta1 stimulated epithelium. Nanotechnology. 2010;21; 265102. 2010;21; Nanotechnology. epithelium. stimulated beta1 - 1572. - 1327. - 396.

The cornified envelope: a model of cell death in the skin. Nat Nat inthe skin. celldeath aof model cornified envelope: The the etiopathogenesis ofAD

lled study. Br J Dermatol. 2007;156; 231 J2007;156; Dermatol. study. Br lled berleithner H, Pavenstaedt H, Riethmuller C. Riethmuller C. H, H,Pavenstaedt berleithner - - 367.

36. possible elicitors - Driven Depletion of Filaggrin Proteins. In: Proteins.In: Filaggrin of Depletion Driven

- 180. antile eczema; useful parameter to to parameter useful eczema; antile

- 901.

for a and other formsother of dermatitis and

like projections of of like projections he UK working party's party's he UK working better understan better Dermatol. 2006;15; 387 2006;15; Dermatol. - 240. Filaggrin Filaggrin A novel A

ding of

- - .

934 2011;66; Allergy. severity. dermatitis andatopic genotype filaggrin both by influenced are products degradation 23 2117 2008;128; Dermatol. JInvest corneum. the stratum factorin moisturizing natural level of to reduced gene lead filaggrin inthe mutations function 22 1094 2013;131; Immunol. Clin skin.J atopic Allergy nonlesional and lesional both in like proteins 21 205 2015;136; signal extracellular (STAT3) and 3 oftranscription andactivator transducer by signal expression filaggrin downregulates 20 131 Sci. 2016;82; JDermatol keratinocytes. human in ERK phosphorylation STAT3 and 19 209 Technol. 2005;11; Res Skin dermatitis. atopic mou the hairless using bases cosmetics of applications topical daily of the efficacy of evaluation 18 765 Res. 1996;288; Arch Dermatol seasons. the ageing and 17 329 2013;22; Dermatol. Exp skin. inhuman envelope cornified ofthe composition inthe changes 16 This article isThis article by protected copyright. All rightsreserved.

Accepted Article - 940. - Kezic S, etal.Loss LE, HB,Campbell Thio Jongh CM, ES,de Koster PM, Kemperman Kezic S, Jean BalicaS, Hsu CY, J, Henry Pellerin L, Ki IL SW. Son HJ, HC,Ryu H, Bae Lee Ryu WI, H.Objective Tagami H, SakaiY, M, Ohshima Oyobikawa K, Mizukoshi K, Matsumoto lipids: corneum Stratum A. Rawlings J,A, Banks Mayo Harding C, Rogers J, M, Rinnerthaler 208 e209. 208 m JH, Bae HC, Ko NY, Lee SH, Jeong SH, Lee H, et al. Thymic stromal lymphopoietin lymphopoietin stromal al.Thymic LeeH,et SH, Jeong KoNY,Lee HC, m JH,Bae

- regulated kinase (ERK) phosphorylation in keratinocy in phosphorylation (ERK) regulated kinase

O'Regan GM, Yau N, Sandilands A, Chen H, Campbell LE, et al. Levels of filaggrin Levels of al. LE,et H,Campbell A,Chen N, Sandilands GM,Yau O'Regan

Duschl J, Steinbacher P, Salzmann M, Bischof J, Schuller M, et al. Age M,et J,Schuller al. Bischof M, P,Salzmann J,Steinbacher Duschl - 2119. - 217.

- Decoster C, Mechin MC, et al. Defects of filaggrin of Defects et MC, al. Mechin Decoster C, - 33 down - 770.

- regulates filaggrin expression by inducing by inducing expression filaggrin regulates tes. J Allergy Clin Immunol. Immunol. Clin JAllergy tes. - 134. the effect of the effect

se model of se model - - - 1102. of 335. - - related related

- dermatitis patients Table 1. This article isThis article by protected copyright. All rightsreserved. b a Highlighted in bold are bold thein resultsHighlighted significant (P<0.05) U²Mann-Whitney test ¹Independentsamples t-test log-transformed with DTIvaluesand of NMF *DTIvalues participantsarefrom 5 missing values participantsare †NMF from missing 17 thefilaggrin,U Mann-Whitney test groups)(2 testor Kruskal-Wallis (>2 groups) wasused R2447X Thegenefilaggrin2282del4, analyzedare:mutations R501X, Carriers Homozygote: double-allele with eithermutations null heterozygous compound orhomozygous Heterozygote:Carriers one genewith functionalfilaggrin copy Carriers type: genefunctionalfilaggrin 2 copiesWild with valuesTEWL high and were Low defined asvalues belowor above the valuemedian participants of all criteria; AU,diagnostic DTI,Party's arbitrary dermatitis the AD,units; Dermaltoatopic accordingUK Texture Working Index; p-value Historyfissures of on hands and feet p-value Palmarhyperlinearity p-value Clinicalxerosis p-value FLG p-value FLG p-value HistoryAD of p-value Keratosispilaris p-value TEWL(g/m p-value Historyasthma of or hay fever p-value group Age p-value Sex Overall Independentsamples t-test groups)(2 (>2or One-waygroups) ANOVA log-transformed with values wasused testofto DTI, the NMF difference andfilaggrinstatistical characteristics population thecontrolsstudy tohealthy accordingwithin Independentsamples t-test groups)(2 (>2or One-waygroups) ANOVA log-transformed with DTIvaluesand was used testto of NMF thedifference statistical characteristics population thestudy topatient accordingwithin population.For mutations mutations 2 Accepted Article )/h) Heterozygote

Homozygote 30-50 years 30-50 The DTI, level The DTI,level of High ( High Low (<12) Low Wild type Wild >50 years >50 years <30 Female Male ≥ Yes Yes Yes Yes Yes Yes 12) Yes No No No No No No No Patientpopulation 48.9 (23/47) 48.9 (16/47) 34.0 (29/47) 61.7 (18/47) 38.3 44.7 (21/47) 44.7 (26/47) 55.3 (30/47) 63.8 (17/47) 36.2 (14/47) 29.8 (33/47) 70.2 (20/47) 42.6 (19/47) 40.4 (28/47) 59.6 (19/47) 40.4 (37/47) 78.7 (10/47) 21.3 (31/47) 66.0 (16/47) 34.0 (27/47) 57.4 (20/47) 42.6 (29/47) 61.7 (18/47) 38.3 %(n/ntotal) 17.0 (8/47) 17.0 (8/47) 17.0

and healthy controls a 75.0 (15/20) 75.0 (13/20) 65.0 (17/20) 85.0 (11/20) 55.0 (10/20) 50.0 (10/20) 50.0 %(n/ntotal) 25.0 (5/20) 25.0 (7/20) 35.0 (3/20) 15.0 (5/20) 25.0 (4/20) 20.0 Controls • • • • • • • • • • • • • b monomeric filaggrin Patientpopulation 82 (63/110) 82 (51/108) 75 (43/120) 67 (57/138) 71 (62/108) 83 (62/108) 83 (51/108) 78 (63/105) 86 (41/118) 79 (48/134) 73 (55/114) 86 (60/150) 85 64 (48/97) 64 (60/90) 74 (59/98) 77 (39/97) 67 (49/98) 67 (60/87) 67 (47/93) 67 (59/90) 69 (62/92) 75 (59/87) 67 (51/82) 67 (47/98) 70 (59/98) 75 0.336 0.979 0.519 0.170 0.556 0.786 0.310 0.700 0.191 0.553 0.197 median (25th/75th percentile) (25th/75th median 114 (93/139) 114 45 (31/109) 45 (33/100) 47 (32/109) 43 44 (29/47) 44 (37/87) 47 (29/88) 40 (25/63) 45 (29/44) 33 (30/59) 44 (31/78) 44 30 (20/ 30 <0.0001 Controls 0.271 0.701 0.107 0.508 DTI* • • • • • • • • • • • • • , stratified bystudypopulation characteristics. •) PatientControls vs. p-value 0.030 0.012 0.025 0.023 0.025 0.049 0.015 0.074 0.219 0.055 0.392 0.090 • • • • • • • • • • • • • ¹

(AU/mg epidermis) and 100.2 (23.8/172.2) 100.2 (27.6/172.2) 100.2 Patientpopulation 127.4 (40.0/370.9) 127.4 (41.1/351.7) 150.5 (41.1/351.7) 150.5 FLG, 69.7 (23.1/180.5) 69.7 (10.3/175.6) 31.8 (13.7/150.5) 58.7 (18.1/183.1) 51.7 (23.1/127.5) 51.7 (13.7/180.5) 44.1 (12.6/227.1) 49.5 (19.8/153.0) 44.1 (11.3/193.8) 64.2 (16.5/180.5) 58.7 65.9 (23.1/206.6) 65.9 (26.4/195.8) 69.7 (23.1/134.7) 64.5 39.1 (1.3/159.9) 39.1 (3.5/130.9) 26.3 (2.5/116.1) 23.6 (9.0/187.8) 44.1 (9.0/209.1) 58.7 8.8 (0.4/134.3) 8.8 0.4 (0.3/1.7) 0.4 <0.0001 <0.0001 filaggrin gene mutation; NMF, naturalmoisturizingfactors; TEWL,transepidermalNMF, gene filaggrin mutation; water loss 0.004 0.008 0.140 0.659 0.559 0.914 0.526 0.812 0.793 median (25th/75th percentile) (25th/75th median

Filaggrin (AU/mg epidermis)Filaggrin (AU/mg 211.6 (124.6/537.7) 211.6 (187.0/523.9) 211.6 (113.6/448.8) 187.0 (128.1/280.4) 156.6 (102.7/523.9) 211.6 (102.9/388.3) 207.5 (108.1/486.3) 195.2 125.0 (69.7/363.6) 125.0 (81.4/409.6) 131.6 (40.2/900.0) 460.7 (93.9/527.3) 171.8 211.6 (36.8/ 211.6 Controls 0.392 0.347 0.956 0.980 0.775 • • • • • • • • • • • • • •) PatientControls vs. <0.0001 p-value 0.003 0.010 0.039 0.026 0.015 0.208 0.232 0.118 0.116 0.061 0.001 • • • • • • • • • • • • • NMF (mmol/g protein) NMF (mmol/g protein) ² Patientpopulation 0.63 (0.47/0.83) 0.63 (0.46/1.05) 0.72 (0.43/0.94) 0.64 (0.51/0.97) 0.75 (0.28/0.72) 0.46 (0.55/0.99) 0.70 (0.25/0.44) 0.32 (0.59/1.01) 0.72 (0.47/0.95) 0.73 (0.46/1.00) 0.64 (0.47/0.95) 0.73 (0.41/0.80) 0.63 (0.66/1.19) 0.94 (0.41/0.95) 0.65 (0.47/0.99) 0.67 (0.43/0.92) 0.63 (0.61/1.02) 0.72 (0.39/0.78) 0.64 (0.58/1.10) 0.81 (0.83/1.28) 0.99 (0.40/1.01) 0.72 (0.46/0.69) 0.59 (0.43/0.99) 0.67 (0.47/0.89) 0.64 (0.47/0.96) 0.66 (n=47) 0.414 0.709 0.100 0.006 0.872 0.027 0.836 0.327 0.020 0.023 0.905 median (25th/75th percentile) (25th/75th median NMF (mmol/g protein)†(mmol/g NMF 0.92 (0.61/1.08) 0.92 (0.81/1.76) 1.05 (0.44/1.07) 0.81 (0.90/1.78) 1.05 (0.77/1.47) 1.04 (0.87/1.60) 1.09 (0.82/1.11) 0.99 (0.89/1.80) 1.05 (0.45/1.05) 0.77 (0.78/1.17) 1.01 0.99 (0.86/ 0.99 (0.39/ 0.77

Controls 0.312 0.033 0.924 0.679 0.029 n=20) • • • • • • • • • • • • • •) •) PatientControls vs. p-value 0.025 0.009 0.002 0.001 0.005 0.285 0.413 0.062 0.246 0.564 0.052 0.476 • • • • • • • • • • • • • in ¹

Index circularstru count (AFM). (bottom)basal face of adh corneocytes Figure 1 This article isThis article by protected copyright. All rightsreserved.

Accepted Article ( DTI). DTIisdef B) . Nanostructure counting.

Arbitrarily addressed areasArbitrarily addressed of20µm ctures spots). (green Thenumber ofobjects Dermal areayieldsthe per Texture ined asmeanined of10 images. value A)

Su ering ering to theisimaged tape byAtomic M Force perficial ²

are scannedare andanalyzed visionto bycomputer corneocytes obtainedbytapestripping. The are

AD, atopic dermatitis AD, atopic

icroscopy icroscopy Texture Index; NMF,naturalmoisturizing factors used was test statistical differenc DTI andNMF,i in dermatitis patients and healthy controls expressed median as with values interquartile range. Figure This article isThis article by protected copyright. All rightsreserved. P groups. way ANOVAfollowedtestwas byTukeyposthoc to statistical used test the difference betweenthe Figure 3.

D e rm a l T e x tu re In d e x (D T I) D e rm a l T e x tu re In d e x (D T I)

Accepted Article1 1 5 0 5 0 0 0 0 1 1 2 2 5 0 5 0 . 0 0 0 0 0 -

The DTI The DTIinhealthyparticipantsstratified group, asmean byage expressed SD).One (± values <0.05 significant.were considered DTI,Derm P

< a . P t

3 i 0 e n y t s e - ndependent samples t P a <0.05werevalues significant.considered r = s 0 . H 0 , e betweenthetwo groups 1 e 5 a levels of l t h y

3 n

0 c s - o

5 n P

0 t r < y o l 0

e s a .

r 0 s 0 monomeric filaggrin 0 P 1 < 0 .

> 0

5 0 0 y 0 F ila g g rin p ro te in (A U /m g e p id e rm is ) 1

e - a test with log transformed values r 2 4 6 s 0 0 0 0 0 0 0 , while for, while P a t i e n t

P (AU/mg epidermis) < 0 H . 0 e 0 1 a l t h y monomeric

c o

n t r AU, arbitrary AU, DTI, arbitrary units; Dermal o l s al Texture Index Texture al filaggrin the Mann

T o ta l N M F m m o l/g p ro te in and NMF were used to were used test the 0 1 1 0 . . . . 5 0 5 0 P a t i e

n (mmol/g protein)(mmol/g t s P = 0 . H 0 0 e 5 a - l t Whitney U h y

c o n t r o l s For -