This article isprotected rights by All copyright. reserved. 10.1111/exd.13867 doi: lead todifferences version between this andthe ofPleasec Version Record. been and throughtypesetting, proofreadingwhich may thecopyediting, process, pagination This article undergone has for and buthas accepted been not review publication fullpeer 4 Germany; Berlin, 3 Universitätsmedizin, 2 Hungary 1 Articletype : Regular Article RALPHDR. RÜHL (Orcid ID 0000: docosanoid signalling docosanoid

Department of Biochemistry and Molecular Biology, University of Debrece of University Biology, Molecular and Bi Paprika Biochemistry of Department Department of Dermatology, Faculty of Medicine, University of Debrecen, Debrecen, Debrecen, Debrecen, of University Medicine, of Faculty Dermatology, of Department Transcriptomic and and Transcriptomic

AcceptedAllergy Article DanielTöröcsik RenataLucas

- Center o analytics BT, Debrecen, Hungary Debrecen, BT, analytics - of hrt, eatet f Dermat of Department Charité, human 1, 1

,

C JohannaMihaly hristin Weise

- 0002 atopic atopic lipidomic -

8410 in in

- affected and non and affected 6659) 2 , dermatitis . 3

JanineGericke

,

MargittaWorm

profiling ology and Allergology, Charité Charité Allergology, and ology

patients 3

, of eicosanoid / / eicosanoid of

Andrea Szegedi 2 , - Ralph affected affected n ite this articleite this as ; ;

Hungary;

Rühl 3 , skin 4

1

, –

This article isprotected rights by All copyright. reserved. affected andpar pro a hallmarkof a parametersthese are All m non in higher evenwere lower were patients AD of serum the in as pro know are which COX were metabolites DHA and EPA AA, of sum the non non and pathways COX various of expression non and affected as well as serum as pro protectins. bio highly to PUFA for enzymes main the are (COX) cycloxygenase and (LOX) Lipoxygenases Abstract E Tel: +36 H Mezögazdászutca 62 Paprika Bioan Dr. Ralph Rühl Corresponding author: - - ainly upregulated ainly mail: r

Accepted Article400 - - tpc d atopic - inflammatory vs pro vs inflammatory resolving 1 and ALOX12B expression ALOX12B and 1 fetd kn an skin affected 2 Debrecen - alphruehl - affected sk affected 30 2330 501

LOX and COX pathways are impor are pathways COX and LOX ermatitis in in

alytics BT - eaoie ytei ad eaoim o vros inflammatory various for metabolism and synthesis metabolite cie rsalnis luoree, hobxns l thromboxanes, leukotrienes, prostaglandins, active

. Decreased . non n tly intly non

@ - o nue th ( itch induce to , affected and affected AD affected and affected web.de

in were in

while srm Mono serum. d

A) I ti suy e determined we study this In (AD).

nye, idn poen ad eetr involve receptors and proteins binding enzymes,

- - affec lipid resolving

-

affected skin ofAD n3

EPA and DHA levels in ser in levels DHA and EPA found; /n6 ted AD ted , -

PUFA ee increased were COX 12

- in addition n3 addition in affected skin samples from AD from samples skin affected - - - HETE, yrxltd PUFA hydroxylated skin. To conclude, 12/15conclude, To skin.

and a mediators in non in mediators nd

12/15 metabolite - - T4 TB,PE ad PGF2) and PGE2 TXB2, LTB4, inflammatory and non andinflammatory s - tant for the regulation of pro of regulation the for tant patients. kin. Expression of COX1 and COX1 of Expression kin. increased in affe in increased , /n6 -

LOX hl n3/n6 while - PUFA ratios were lower in affected and affected in lower were ratios PUFA

um and reduced EPA level in affected in level EPA reduced and um

ratios were lower in in lower were ratios metabolites

- - eaoie o A, EPA AA, of metabolites affected and affected skin as well well as skin affected and affected

UA ad PUFA and PUFAs - - LOX and COX pathways werepathways COX and LOX PUFA cted and non and cted - - as well as various lipids, various as well as resolving environmentresolvingin patients and the and patients s ipoxines, resolvins and and resolvins ipoxines,

n mtblt ratios metabolite and i tee O and LOX these in d

AD - COX n te ai of ratio the and inflammatory - eaoie in metabolites - - diseases affected skin. affected patients - - metabolites metabolism DA and DHA ,

dermal

skin such or .

This article isprotected rights by All copyright. reserved. mediatedsignaling andFABP7 with attraction of DHA seem FABP4 acids. fatty various of attractors and transporters traffi processes immunoregulatory and differentiation gland sebaceous in involved barri epidermal of maturation and p (LXA4R receptor 2 and (TXB2 receptor thromboxane ( B4 d eicosanoids/ during synthesis docosanoid synthase thromboxane l resolvins (lipoxins, pro with acid eicosapentaenoic AD in responses docosanoids and P po in pivotal function eosinophils and lymphocytes background dryness chronicdermatitisis a (AD)Atopic Introduction pxgnss (LOX) ipoxygenases eroxisome prolifera eroxisome olyunsaturatedacidsfatty

Accepted Articlelymorphism eicosanoidsdocosanoids / c LTB4 k ), n i mdae va f via mediated is ing , s pr

- ) eczem n IE eitd mechanisms mediated IgE and nlmaoy (prostaglandins, inflammatory ostaglandin E2 ( E2 ostaglandin

tp development atopy

eetr (L1 n 2 and (BLT1 receptors disturbed s

ocosanoidsGPCRsbe potentactivatorstowereshownof as tu si lesions skin atous

a well as ) (reviewed in (reviewed

reviewedin r ivle i t in involved are protecti , tor

skinbarrier pathways (EPA

- activa and

PG ) (PUFA) s ciaos of activators as ,

are , rsalni recepto prostaglandin ), leukotriene

ted receptors ted

E2

a r n sbct activation sebocyte and er nlmaoy condi inflammatory 5 4, 3 edn t a to leading s maresins ns, 11, 12 11, , , t ai bnig rtis (FABP proteins binding acid tty

) also pointed )

, the major the studies n frhr e further and . E1 2 3ad 4), and 3 2, (EP1,

and relapsingand

altered . PP , and A , cysteinyl ), ra e oiiain of modification he rrtc excoriations pruritic ARα plays an importantrole during skin development, chidonic acid chidonic especially their bioactive metaboliteseicosanoidsbioactiveespeciallythetheir - syn

confirming immuneresponse

l pa a role a play all ektins trmoae) or thromboxanes) leukotrienes, ( h / h shift, Th2 / Th1 precursors of a large variety of lipid mediators lipid of variety large a of precursors thases PPARα and PPARγ and PPARα ) on ula hroe receptors hormone nuclear -

activity nzymes leukotrie the importance ofgenetic factors inflammatory in o alri diseases allergic of tions

r involved are

(AA 18, 19 18, s for rs h association the rsalni F ( F2 prostaglandin

) e eetr (CYS receptors ne , docosahexaenoic acid (DHA) acid docosahexaenoic , . uh as such 6 1, aiu apcs of aspects various C .

3 14 13, 2 and .

rsalni D ( D2 prostaglandin of eet i preblt barrier permeability in defects Although s skin

)

yclooxygenase (COX) and and (COX) yclooxygenase

v while ,

to be associated with PPAR with associated be to ar lichenification are activated by fatty acids fatty by activated are )

which , n iatv ecsni / eicosanoid bioactive in

ious immunecellsious disease prostaglandin

f AD of , PPARγ seems to be be to seems PPARγ xenl factors external PG suchas

, characterizedby, F2 , 6 4, r intracellular are L

TR , 7 5, )

. inflammatory pro with , 2 1,

PGD2 F) lipoxin (FP), 15

- - leukotriene Mainly 10 . - 2 and 1 synthases

17 - Various .

. resolving

. ,

various n the In ) Ligand mainly

(DP1

and and are are the ), - ,

This article isprotected rights by All copyright. reserved. Accepted (EA1/168/06) committee ethics local the from obtained was at stored and portions µl 250 to aliquoted was serum The 4°C. and g 1500 at down spun and temperature room at minutes 70 an patients same the from drawn were samples serum phenotype extrinsic / intrinsic combined of patient one and extrinsic of are 42.5, limited to due included, were patients 3 at stored and ice dry non of affected of biopsy one patients, AD of case Indisease. atopic of history female individuals; average age declaratio the patientswithsix AD (twomalefour female and a involved in consent informed obtaining after taken were specimens biopsy Skin ArticleSample collection Materials and methods receptors further dermatitis atopic in altered is signaling docosanoid / eicosanoid micro analyzing non levels docosanoid bi and receptor we study this In ° - C until analysis. until C affected skin of AD patients. AD of skin affected average total IgE averagetotal - affected skin were taken from each patient. Specimens were immediately frozen in frozen immediately were Specimens patient. each from taken were skin affected plan and/

targeted therapy targeted . n of Helsinki, and from six non six from and Helsinki, of n

- or enzymatic inhibitors ofvarious mut o aalbe kn biopsies. skin available of amounts dn poen expression protein nding using investigate

For preparation of the serum samples blood was allowed to allowed was blood samples serum the of preparation For - 70

4126 lipidomic techniques in the skin of healthy volunteers and volunteershealthy of skin the intechniques lipidomic

° C

until RNA isolation or HPLC analysis. HPLC or isolation RNA until KU/L and specific IgEspecific and KU/L d for atopic dermatitis atopic for

eiconsanoid 30 years) characterized the by absence ofpersonal orfamily - 80°C unt 80°C We used an used We vial si tsu (ave tissue skin available il further analysis. further il -

using - docosanoid / atopic healthy volunteers (two male and four and male (two volunteers healthy atopic

d applied an applied d patients; averageyears;patients; age 31 f h Uiest o Dbee i Hungary in Debrecen of University the of involvedenzymes.

transcriptomic 49 using selected agonists / antagonists for antagonists / agonists selected using u am was aim Our

KU/L, d healthy controls and were kept at kept were and controls healthy d

- eitd signaling mediated

optimized lipidomic optimized 1 male / 2 female2 / male1 Ethical approval for the study the for approval Ethical and For HPLC For s el s eicosanoi as well as o get to

ae g 2, SCRORAD 28, age rage based on our results our on based skin and one and skin

an - MS analysis just analysis MS

). vriw how overview , two patientstwo ,

i enzyme, via

accordingto Additionally, method for method

clot for 10 for clot reas from reas affected

biopsy biopsy / d to to / -

This article isprotected rights by All copyright. reserved. Accepted microscope light Axioplan the using magnification equipped with aAxioCamHR andAxiov x100 at taken Darmstadt, were (Merck, images Finally, counterstaining hematoxylin by and (DAKO) Phosphatase/RED antibody. temperature. room at min 30 for Hamburg, anti goat biotinylated UK Oxfordshire, (Abgent, The tissue cryo Immunohistochemistry Article and analysis, relative foldinduction was data determined by the compar for utilized was Biosystems) (Applied 2.1) (version Software Detector mRNA A cyclophilin to normalized methods curve standard or CT comparative H)Budapest, pre System Synthesis Standard First QRT H). Budapest, Kft., Magyarország Technologies II SuperScript the using cDNA into electrophoresis.realFor Scien (Thermo spectrop NanoDrop of means by measured were RNA of purity and concentration beads metal O Cincinnati, Inc., Center Research (Molecular homogenized were samples Skin Analysis of mRNA expression - eind G asy odrd rm ple Biosystem Applied from ordered assays MGB designed D eeto wa Detection ).

n ttl N ws sltd codn t te auatrrs udlns The guidelines. manufacturer’s the to according isolated was RNA total and

using Nonspe tific, Bioscience, Budapest, H Budapest, Bioscience, tific, - sections (5 µm) were incubatedwith antibodies againstthe human ALOX12B

an ABI Prism 7900. Relative mRNA levels w levelsmRNA Relative7900. Prism ABI an - cific binding was blocked with 5 with blocked was binding cific rabbit IgG rabbit - time quantitativetime(QRT PCR promd sn Dk RA™ eeto Sse Alkaline System Detection REAL™ Dako using performed s o COX or )

and anti and

Negative controls were performed by omitting the primary the omitting by performed were controls Negative (F/4 Acm l, abig, K, olwd by followed UK), Cambridge, plc, Abcam (5F6/F4, 1 ih IGN ise Lyser Tissue QIAGEN with ision analysis software (Carl Zeiss, Jena - mouse IgG, respectively (both DAKO Diagnostika, DAKO (both respectively IgG, mouse ). RNA quality was checked using agarose gel agarose using checked was quality RNA ). H , USA) , - - ative threshold cycle method. using triplicates in out carried was PCR reversetotalwasPCR) RNAtranscribed % bovine serum albumin (BSA) in PBS in (BSA)albumin serum bovine %

using previously autoclaved QIAGEN autoclaved previously using

(ple Boytm, Applera Biosystems, (Applied s ere calculated ere n r raet solu reagent Tri in

Ivtoe, Life (Invitrogen, with , D ). .

hotometer

either theeither Sequence

tion D ). - , This article isprotected rights by All copyright. reserved. MassLynx the via controlledsoftware (Waters was system The detector. a as UK) Waters, from PT Ultima MS A compartment. (Waters module system Chromatographic immediatelyanaly pre the in placed (Waters sample septa silicone / PTFE with vials top screw brown into transferred micro into H (Fluka, Sigma from Plus The docosanoids. and 25 approximately with resuspended eicosanoids of degradation prevent with vented was concentrator Eppendorf The µl. ~10 was volume Eppe anreactionvials with Eppendorf HODE 15 ( containing 130 min, were added mg 50 to added waterwaspresent, was biopsyskin of mg 50 that less (ifbiopsy skin a of mg established preparation Sample minor modifications HPLC the for procedure described previously The skin andserum HPLC

Accepted - Article hydroxy shaken for 3 min, the precipitated protein was centrifuged at 13.000 rpm, 4°C for 6 for 4°C rpm, 13.000 at centrifuged was protein precipitated the min, 3 for shaken ) -

ESI and of (D4) )] to yield to )] - μ sample weight sample - injectio - o te eutn spraat a sie wt 10 with spiked was supernatant resulting the of L the MS method used for retinoid quantification retinoid for used method ioaereoc cd ( acid eicosatetraenoic prostaglandin D2 ( D2 prostaglandin ec a a ocnrto o 1 of concentration a at each , - skin samples were cut with scissors with cut were samples skin , H , MS analysis of free fatty acids and eicosanoids and docosanoids in docosanoids and eicosanoids and acids fatty free of analysis MS - , H

- Aldrich, H Aldrich, sed cooled (15°C) autosampler of the Waters 2695XE separation module separation 2695XE Waters the of autosampler (15°C) cooled vi inserts n ) including a gradient pump, autosampler, degasser and a heated columnheated a andautosampler,degasserpump, gradient includinga )

). - 7, 20, 21 20, 7, exactly MS detector with an ESI ionizing option was used (Micromass Quat (Micromass used was option ionizing ESI an with detector MS .

. The whole analytical sample preparation procedure is based on an on based is procedure preparation sample analytical whole The .

. for our standardized extraction protocol extraction standardized our for

) and 35.5% a 35.5% and ) 35 Te PC ytm osse o a aes 65E separation 2695XE Waters a of consisted system HPLC The . als (Waters, H (Waters, als PGD2 μ l, then vortexed (15 sec), shaken (3 (3 shaken sec), (15 vortexed then l, 15 ) μ

- nd l of H of l (D4), HETE orf concentrator at 30°Cfor concentratorat orf cetonitrile (Merck KGaA, D KGaA, (Merck cetonitrile PLC solvent A [64.3% water (water, Chromasolv (water, water [64.3% A solvent PLC μ/l s h itra control) internal the as μg/ml 0 ) ). These glass vials with the 35 the with vials glass These ). 5

- D) and (D8) hy droxy - 22 ESI in sm in . In summary, to 50 to summary, In . - eicosatetraenoic acid ( acid eicosatetraenoic -

9 MS - hydroxy all pieces all - S ehd a followed was method MS μ iooe uentn mix supernatant isotope l - ) semi caeaini ai ( acid octadecadienoic , ) and ) samplethe untilmin 60 on ice on 150 ihy pure highly min) and transferred and min) - re etat was extract dried μ μ l acetonitrile was was acetonitrile l 0.2% formic acid formic 0.2% . These mixtures These . l of serum or 50 or serum of l eaoae in evaporated , μ 5 l extract were extract l - HETE ro to argon H , )

and )

(D8),

yield with and and tro 9 -

This article isprotected rights by All copyright. reserved. as supplementary table 1. well as coefficient, correlation recoveries segments, time individual method, used energy, quantification collision eicosanoids/docosanoids times, PUFA, retention for settings monitoring reaction Multiple MS resolution 14.5; HM2 resolution 14.5; the Ion ene at substance each for set are parameters entrance 0.7; 1 energy Ion 14.5; resolution HM1 14.5; resolution (fo V 0.2 and 1) (for V 35 at set was voltage lens RF 3 was current capillarythe l/h, 10 was flow gas cone the 400°C, was temperaturedesolvation the l/h, 780 the eluent HPLC following the ionsource temperature of85°C.The des Multiple reaction monitoringsettings: with ESI, a negative ESI 3.6at e Scientific source, ionization spray (electro ESI used. Waters, was bar 0.8 of pressure inlet an with Argon options. MS of MS different two and conditions HPLC same the using twice performed was step This used. was analysis From 40°C. to heated was column the and ml/min0.4 adjusted to was rate flow The B. % 5 min16.0 and B % 100 min15.9 B, % 100 ( B solvent min15.0 B, % min605.0 B, % 20min B, 3.0 20%min following 0.0thesteps: of consisted and above) (see A solvent from ( KgaA Merck RP 100, Superspher mm; 2 X 125 (LiChroCART, column analytical the in an through passed then mixing, to prior conditions. HPLC

Accepted Articlea larger

- 3 mbar. H

NM30 Ni NM30 ) was vented by nitrogen continuously produced by the nitrogen generator (Peak generator nitrogen the by produced continuously nitrogen by vented was )

varietyof - MS analysis optionsanalysis MS D The Micromas The μ ) embedded in the column compartment. A multilinear gradient was formed was gradient multilinear A compartment. column the in embedded ) A, and the cone voltage was 50 V. Aperture voltage was set at 0 V and the and V 0 at set was voltage Aperture V. 50 was voltage cone the and A,

trogen generator) including compressor (Waters compressorincluding generator) trogen

h euns ee easd n h Wtr 29X sprto module separation 2695XE Waters the in degassed were eluents The analytes.

s Quattro Ultima PT was controlled via the MassLynx software. MassLynx the via controlled was PT Ultima Quattro s

(method A or B) or A (method int er -

a and day the same biol same the - line filter (1 filter line rgy 25.0 anda multiplier energyof 650 V. ehnl Me methanol; for optimized – intra LM1 were settings analyzer The 2). r

- ehd aaees; xt ; LM2 2; exit parameters); method ogical extract, 11 extract, ogical 2 - μ day m; Knauer, D Knauer, m; – c Ka D KGaA rck

resolution and quantificationresolutionand setting,was performedwith - , H , variations – ) with the inlet the with ) 1; collision 0 (collision 0 collision 1; - olvation gasflow was 18, endcapped) from from endcapped) 18,

) before reaching before ) μ . h gradient The ). l for each HPLC each for l r lse in listed are

flow set flow

,

This article isprotected rights by All copyright. reserved. were levels non and affected as well as healthy from skin in PUFAs n Increased Results fortest independentsamples significantbe to wasconsidered<0.05 of value P USA).A IL, Chicago, Inc., subjects.6 for stands a ratios as well as statistics of calculation further ( ng/ml point. data per values LC Statistics analysis adjusted andcalculated was timesretention and standardcompounds were used extraction optimal ensure To compounds. “Are the of determination Quantification. reference eicosanoids PUFAs, and docosanoids were usedfor the assay validation. 10 of concentration final Charles Dr. and S) (Malmö, Internationa obtaine solutions the dissolving by prepared solutions. Standard 3

Accepted- Article /n6 MS

- data are shown as as shown are data or . PUFA

g)) were replaced with a value of half of the detection limit (0.1 ng/ml ( ng/ml (0.1 limit detection the of half of value a with replaced were g))

AA increased (Kastel l

ratios levelsaff in niiul ioaod ad ooaod wr qatfe bsd n the on based quantified were docosanoids and eicosanoids Individual

and extraction efficiency based on an average of all four added isotopesaddedfour all averageof an on efficiencybasedextraction and - e KT Bdps, ) Sigma H), Budapest, KFT, Med

Statistical analysis was performed using the program SPSS 16.0 (SPSS16.0program SPSStheperformedStatistical using wasanalysis in

in tc sltos f h PFs ecsnis n dcsnis were docosanoids and eicosanoids PUFAs, the of solutions Stock aus hc wr dtrie udr h qatfcto lmt (0.2 limit quantification the under determined were which Values non a Under the Curve” (AUC) and compared with the AUC of standard of AUCthe with compared and (AUC) Curve” the Under a kn of skin μ mean of mean g/ml. All stock solutions were stored in darkness at darkness in stored were solutions stock All g/ml. . This analytical procedure was established for liquids and tissueand liquids establishedfor wasanalytical procedure This . Serhan (Harvard Serhan - ected and non and ected .

affected

( PGD2 (D4),5 AD three individual three - (2 patient .0 -

fold) n peiin o quantification for precision and d from Cayman from d -

University affected - HETE (D8),15 HETE s : and affected and ntal, e xmnd h lvl of levels the examined we Initially, nd sums. For PCR For sums. nd measurements - lrc (Budapest Aldrich

, skin of skin

MD, - fetd kn f AD of skin affected - Chemicals ( Chemicals

- USA) with USA) skin HETE (D8)HETE and9 AD

(1.8 and and - patients

data - standard error mean error standard , fold)

Tallin, methanol to yield a yield to methanol ) Lrdn Lipids Larodan H), each data each

using stp labelled isotope of

and - - EST patien AD HODE (D4)) -

80°C. The The 80°C. student t student - or reduced ), BioMol ), patients g)) for g)) ts. ts. point free free AA s - ,

This article isprotected rights by All copyright. reserved. met AA ev further To serum decreased metabolites skin affected which t on focussed we of concentrations Increased decreasedin non in reduced in decreased slightly and patients r The (0.3 affected DHA of metabolites skin affected metabolites skin affected the on of levels Increased af 1 Figure of levels serum and comparable while fected and

Accepted- Article eaoie. h ra The metabolites. h abolites - hydroxy ydroxy fold) to of atios no alteration was observed was alteration no

and w . Ratios ). ere (1.8

in AD were - DA were /DHA (0.3

- of EPA of PUFAs, from metabolites the ioaereoc cd ( acids eicosatetraenoic - affected skin aswe serum both aluate the ratios between the PUFAs the between ratios the aluate (4.5 fold) (AA (1.6 EE/EE were HEPEs/HETEs - - -

serum fold) fetd AD affected increased he sum of sum he eu lvl wr increased were levels serum -

- met - fold) of , the , nrae in increased fold) n afce skin affected and the , of AD .

n3/n6 2.9 : . PUFA EPA

of hydroxy nrae i afce skin affected in increased f AD of - hydroxy is f AA of tios patients AD (2.2 -

- - od DHA fold; AA metabolites PUFA - - ains skin patients - h patients as patients - - - metabolites ydroxy fold) ll asin serum of AD ains and patients - eicosapentaenoic acids ( acids eicosapentaenoic non PUFA -

in ooaeani ais ( acids docosahexaenoic DHA uh as such (Figure 2 reduced AD HETEs - in affected skin ofAD AD affected - (6.5 e on ta te l the that found we met -

- - - pati met - w metabolites metabolites patients (EPA

r lwr n AD in lower ere - 4.0 : well as increased in AD in increased as well

fold) ) ents abolites (AA and ) , EPA/ in

.

w -

oprbe n AD in comparable met) both both (AA - ere - (1.7 met) fold) f AD of remain serum AA serum

, - A, EPA /AA,

nrae in increased - met - we calculated ratios of EPA of ratios calculated we patients(F while , and non fold) remain as well as DHA as well as

of AD of

- in n h si o AD of skin the in . patients 3.1 : ed .

- ais o Ratios EPA levels in skin and serum were serum and skin in levels EPA affected HEPEs DHA/ h AD the

- - and evel patie nhne i afce si and skin affected in unchanged HDHAs patient - ed ains wie ut for just while patients, - od DHA fold; AA -

met s AA igure 2 , DHA

)

nts oprbe n AD in comparable - , non hl they while of metabolites/AA - HDHAs/ f

- ains serum patients ) were serum s - abolites n afce si of skin affected and patients were , . serum

AA - - - ee incr were eaoie were metabolites affected metabolites ). -

hyd

just increased in the the in increased just - met also

EA n DHA and /EPA (2

EE were HETEs - r

Tbe ad 2, and 1 (Table oxy patients ee decrease were - 1.8 : skin fold) ae in eased in lower (2.5 - - metabolites, , DHA , Hydroxy . (DHA . : n DHA and - - fold) fold) Hydroxy Focusing - patients th : e - - Next, even met)

non non also AD and a and AA nd d ------

This article isprotected rights by All copyright. reserved. both 12 lipoxygenase (arachidonate d Increased andaffected skin which were skin 8 in AD AD of affectedskin t Surprisingly, fold) in 2 Table Supplementary skin affected in expressed 5 while inflammatory metabolism patients Increased metabolites were lower EPA of metabolites/AA metabolites/AA for metabolites/DHA increased just were levels j were metabolites/DHA ra expression ermal - rae i non in creased

AcceptedHydroxylation Article

andaffected - of AD of infcnl icesd in increased significantly patientsserum LOX - : metabolites/AA

hn ousn o te synthe the on focussing When

- just - 12 levels patients (ALOX5 ciaig rti (LP ad ektin hdoyae LAH wr less were (LTA4H) hydroxylase leukotriene and (FLAP) protein activating he leukotrienes - - / metabolites even increased in affected skin of A of skin affected in increased evenmetabolites metabo

significantly 15 - sum w levels fetd skin affected - (0.2

- ere - pathway patients f 5 of 5 , O pathway LOX profiles (Figure 2 : of

-

even decreased. even fold) ie wr re were lites - Next, we calculated the calculated we Next, (F remainednon in increased ust LOX - - ). the metab lipoxygenase igure 2 .

h e The e The ) vs healthy or non or healthy vs skin increasedin affected 5 which , FA: 0.3 (FLAP: - eaoie increased metabolites

f h 12/15 the of LOX metabolites LOX B) andTable 2 lts remain olites (2.1 non ). of A

peso of xpression

n ALOXE3 and peso o l of xpression i si ad eu o AD of serum and skin in s AA - ue i non in duced - fold) D affected - s h ky enzyme key the is - signi - eaoie/A EPA metabolites/AA, patientsskin.

Ratios of EPA of Ratios - - non od LAH 0.4 LTA4H: fold; eie mtblts n affected in metabolites derived while sis ficantly altered ) . -

affected skin of skin affected

- - ed affectedskin ahas w first we pathways, AO1B 2.5 (ALOX12B: ioyeae w fcse on focussed we lipoxygenases

A

sum of sum aahdnt lpxgns 3 lipoxygenase (arachidonate and LTB4 and CYSTL2

LOX uoree receptors eukotriene - skin fetd kn f AD of skin affected nhne ad DHA and unchanged

5

-

met ( a nt lee in altered not was 8 3.1

- a rdcd in reduced was hydroxylation

in in

(Figure 2 abolites was - - n h snhss of synthesis the in

fold) fold) and - eaoie/P, while metabolites/EPA, fetd n non and affected AD - od AOE: 2.4 ALOXE3: fold; just D even of AD - - patients f AD of /AA patients. - evaluated patients ) significantly .

- significantly - met -

- patients ains wie DHA while patients, pathway - L1 a strongly was BLT1 ains (Figu patients non abolites ( AD Figure 2 Figure The s The - :

metabolites/A 5 - - ) o assess To affected - ains skin, patients , , while, serum kn f AD of skin

l

ipoxygenase

increased in increased mainly which metabolites erum ratios erum

- - decreased and DHA and fold) affected ALOX12B ). Serum ).

e 3, re DHA were (0.

pro and the A 1 ------

This article isprotected rights by All copyright. reserved. affected skin non in decreased AD COX1 was increasedboth in enzyme major the are Cyclooxygenases Upregulated (6.1 w metabolites affected from originating levels Skin ALOX12B protein expression. non vs. affected in infiltration of degree and in 4 (Figure skin AD possesses arather diffuse distribution ofinfiltratingcells. non in pattern perivascular a by characterized non in than affected in distinctive more he the to comparison in prominent was infiltration cell and evident was epidermis of thickening and hyperplasia level protein express gene ALOX12B enhanced The ( AD affected Figure 3, dermal

Accepted- Article patients - fold) feature ) (2.6

, of AD infiltrating cells infiltrating SupplementaryTable 2 with

- s skin - ( (3.6 fold) Fi as sn imnhsohmcl stainings immunohistochemical using cycl d - ue 2 gure strong patients

- a remarkably a significantly fold) AO1B 3.7 (ALOX12B: - ooxygenases ( ooxygenases fetd skin affected n afce skin affected and althy control. Thereby, the histological features of atopic dermatitis areatopicdermatitis of featureshistological the Thereby, control. althy ) of AD staining intensities staining . .

xrsin f rsalni E ytae PE) a strongly was (PGES) synthase E prostaglandin of Expression ( marked by the green arrow). The increased epidermal thickness epidermal increased The arrow). green the by marked non

- 12

patients

increased higher ALOX12B protein expressionALOX12Bprotein higher - - O metaboli LOX affected skin ). - (<0.01 od AOE: 3.0 ALOXE3: fold; COX

( ion in skin of AD of skin in ion (2 Figure 3, Supplementary Table 2 - ) affected skin. The documented dermal infiltration is infiltration dermal documented The skin. affected .1 - oh n non in both - s fold)

mediated

in - fold) o potgadn synthesis prostaglandin for

keratinocytes sm (9.4 - fetd skin affected f AD of - f AD of display fetd kn wees fetd D skin AD affected whereas skin, affected - fold)

Fgr 4 (Figure signaling - - - fold) affected ains wie t a increased was it while patients, - - patients

a

patients was patients d tendency a ed s well as

(marked by the black arrow) and arrow) black the by (marked n oprsn o elh skin healthy to comparison in was . n h si o AD of skin the In ). (1.6 wie h sm f 15 of sum the while , pathway

affected skin affected higher a with associated comparedto - fold) further verified further ). . to

The n afce skin affected and

in AD in nrae in increase

expression

(6.0 health - patients - - patients fold) at the at y skin y - non LOX

of of in in - :

This article isprotected rights by All copyright. reserved. display CD36 and RETSAT 0.2 γ: PPAR fold; of expressionThe PPAR Reduced observed affectedskin in stronger ( ( 2A Figure in displayed ( ( C4 leukotriene we Firstly, activities. mediated calculated and Pro patients t levelssignificantly protein and expression the with agreement In COX1 of percentage the Interestingly, arrow). green the expressinginfiltrating cells seems to be higher in affected than innon by (marked lesions AD in proportion ofinfiltrating cells express path disease in involved cells inflammatory of infiltration massive a AD of skin Lesionalskin. healthy in observed than arrows) black in resulted lesions AD the throughout distributed performe To PD1 ( A4 lipoxin d15d12PGD2

Accepted Article c - onfirm inflammatory vs pro vsinflammatory ) , - target gene expression aei ( maresin .

immunohistochemistry d

in serum of AD xrsin of expression a the LXA4 increased ) remark ,

ucinl lses f eicosanoids of clusters functional nrae C increased hobxn B ( B2 thromboxane MAR) ) , PPARαγ and - od o AD of fold) lipoxin B4 ( B4 lipoxin LTC4 ably

) and Thi . in in - (2.3 ) pat PD, G2 PGF2 PGE2, PGD2, , higher non calculated a ratio versus the sum of pro of sum the versus ratio a calculated p - X gn expression gene OX1 rto a icesd n non in increased was ratio s ed ients resolving lipid mediators lipid resolving rxsm proliferator eroxisome - fold)

-

LXB4 - affected summarised the sum of pro of sum the summarised was significantlywas decreased

tnec o rdcd xrsin n fetd skin affected in expression reduced of tendency a patients. COX1 protein COX1 :

vs. healthy volunteers (Figure 2

using of AD of ) TXB2 ed , wih u t the to due which , resolvin

COX1 thereby augmenting th - (3.6 hl skin whole patients as well as a strong increasestrong a as well as patients ) hl te expression the While

and - fold) fold)

D1 ( D1 expression

5 - lo t h lvl f rti synthesis protein of level the at also dcsnis codn ter biological their according docosanoids / oxo RvD1 n in and

, samples

- - ciae receptors activated 15 ioaereoc acid eicosatetraenoic

) we evaluation, further For . - ,

-

e u of sum he fetd skin affected deoxy e

in the epidermis (marked by the by (marked epidermis the in resolvin D2 ( D2 resolvin in affected skin skin affected ieml hceig f D skin AD of thickening pidermal -

inflammatory Fgr 4 (Figure the

- - patients is characterized by by characterized is patients of A). Δ12,14 affected skin (PPARα: 0.5(PPARα:skin affected

- resolving lipid mediatorslipid resolving ology PPAR

e COX1 protein amount -

affected skin. COX . O1ws broadly was COX1 ). RvD2 ( - 1.5 rsalni D2 prostaglandin - (reviewed in (reviewed (2.1 agt ee like genes target - mediators (LTB4, mediators metabolites - fold) )

(4.8 , - ( PA) and (PPAR) fold) 5 protectin D1 protectin - KETE) - n even and fold)

formed f AD of t , 2 ) was was )

. A . we he as - - ,

This article isprotected rights by All copyright. reserved. non in pathways inflammatory non and w non in higher even partly are which of pathwaysin affected d and eicosanoids various AD binding enzymes, metabolizing docosanoids and eicosanoids these docosanoids / eicosanoids disease skin inflammatory chronic a is dermatitis Atopic Dis reducedexpression target tendency patients decre e The skin skin ofAD 13 and calculateda ( acid octadecadienoic acid eicosatetraenoic ligands 2 in observed was expression expression off ) as .

Accepted- Article n6 cussion: ains oprd o kn f elh volunteers. healthy of skin to compared patients activators PPAR possible of presence the assess To vs healthyvs skin (2.6 the sd o PAα n γ and PPARα for ased - xpression of xpression PUFA - , genes

KODE, while for PGJ2 and PGJ2 for while KODE, .

- eue n3 reduced h epeso o f of expression The fetd kn f AD of skin affected uh as such of reduced expression reduced of - patients(Table 2) -

derived tend

uh as such atty acid bindingproteins (FABPs) wasaltered, not ajust tendencyof reduced ency of increasedencyinlevels the

in affected skin /n6 major d15d12PGJ2, and surprisinglyand

peroxisome proliferator peroxisome ( RETSAT - - 12 13 UA s el as well as PUFA fold) ocosanoids from COX and 5 and COX from ocosanoids - - itch KETE , , 23 8, 7, KODE the .

S n fetd skin affected in ( ty cd idn poen (FABPs) proteins binding acid atty F - ums ofthesewere ligands -

- mediators proteins and receptor and proteins igure affected skin of AD of skin affected patients. fetd kn f AD of skin affected ) and was observed in affected skin of AD of skin affected in observed was - ) , 27

even

. (Figure 3, 1 and rsalni J ( J2 prostaglandin n hs td w md a made we study this In -

5 CD36 partly 2 affected skin of AD of skin affected - E) oxo s w as 4 . - 4

HDHA a significant increase was found in affected affected in found was increase significant a HDHA various various d) uh as such - - , ioaereoc acid eicosatetraenoic hydroxy

affected skin foundPGD2,affected 12 forskin also in non eie FABP4 besides l a epeso o eicosanoid/ of expression as ell Supplementary Table 2 S - activated receptors (PPARs) was significantly was (PPARs) receptors activated PAα 0.5 (PPARα: trong

n3 - L - patients ooaeani acid docosahexaenoic T ly /n6 - - s B4 patients ,

- e observed; We in affected and non and affected in affected AD 12 nrae itch increased

- TB, 12 TXB2, , we , PUFA PGJ2 - just - patients with

- and 15 and

, od PA γ 0.2 γ: PPAR fold;

(Figure 3, (Figure display e R e) . ) significantly higher in a acltd h sm f PPAR of sum the calculated PD, 5 PGD2, , metabolite comprehensive of involvement prominent a

. - (

- - LOX was 15 )A adtoa feature additional An c) skin. EE PE ad PG and PGE2 HETE, dcd PPAR educed ed ) - . - a) patients.

- KETE

Supplementary TableSupplementary eitr ad p and mediators lo a also and 8 and o atrd js a just altered, not tog nrae of increase Strong b) ais n affected in ratios

- - ) I - KETE, affected skin of skin affected ( , ncreased levels KETE, 15 KETE, 4 -

- hyd - 13 fold) HDHA screening of of screening Other edny of tendency docosanoid - - r oxo mediated oxylation 12 ffec f AD of ) PPAR - - . 9,11 - KETE oxo ted ted We F2, ro ------

This article isprotected rights by All copyright. reserved. influ sk also expression 5 the from originating b characterized c phenotype cells inflammatory influxed as well as levels upregulated e that inflammation local of part a not and feature general a is itching non in 37 TXB2 mediators and ALOX12B Surprisingly, presentin AD are cells inflammatory eicosanoid/ eicosanoid/ leukocytes signa pathway i Increased upregulated strongly were enzymes and AD of skin studies Previous a levels LTB4 increased specific skin affected f) signaling

- chronic inflammatory condition 41 Acceptedin Article

xed inflammatoryxed cells (Figure 4 also were highly pr highly were , , , 10 8, 5, 4, ven in the in ven - fetd kn f AD of skin affected

despite the presenceof the despite the major triggers for initiation and maintenance of of maintenance and initiation for triggers major the m , - ooaod eaoiig nye, idn poen ad eetr in receptors and proteins binding enzymes, metabolizing signa docosanoid mediated docosanoid patients famtr sgaig y eue o icesd ret increased or reduced by signaling nflammatory . ould

e 10 COX1 O1 n ALOX12 and COX1 ling peso o xpression y , confirm

andreviewed in cohgs n lmhcts thereby lymphocytes and acrophages LTB4

y ou by non diinl rsne of presence additional

e bevd eas ms lkl this likely most because observed be in the skin the in esent and expressed in affected in expressed and esent 29 of 28 - were 35 - . various - affected skin an inflammatory background is present is background inflammatory an skin affected , . LOX

gop dtrie srn rdcd eiod eitd signal mediated retinoid reduced strong determined groups r It was also observed in this previous study, that selected receptors selected that study, previous this in observed also was It ed 12 I nflux

- strongly an increased pr increased an HETE, PG HETE, pro f pathway - patient eicosanoids and docosanoids and eicosanoids

is of is of inflammatory cells inflammatory of

2, 36 2, - in increased PPAR increased nlmaoy eicosanoid/ inflammatory were

increased in non in increased high atopic dermatitis. s

. , E2 and E2

spotn te lncl idns ht n D patients AD in that findings clinical the supporting , such as such

42, 43 42,

importance . any rsn i krtncts i keratinocytes in present mainly

tog pro strong ). esence ling even n h non the In

PG

LTB4 .

F2 These results altogether confirm and support and confirm altogether results These n h non the in u t hg exp high to due - l of inflammatory cells inflammatory of a

i gands in affected skin of AD of skin affected in gands (Table as well s -

- and even highe even and and influx of inflammatory of influx and affected skin of AD of skin affected in inflammatory

- fetd kn n skin, affected affected and non and affected

2) - and involved and

fetd kn f D patien AD of skin affected ooaod ytei enzymes synthesis docosanoid . the major itch major the skin The i The

. specific inoid the ncreased COX1 and BLT and COX1 ncreased ii mediators lipid Our results also revealed also results Our result r expressed r eso o various of ression chr - even in non in even skin o patients. Major i Major patients. - eetr mediated receptor synthetic affected skin areas skin affected nc inflammat onic te skin the n - AD receptor BLT1 receptor

as n increased in - phenotype is is phenotype

-

cells patients specific indicated by indicated and present and

especially pathways - affected

such as such and n in ing these ts

tch and AD ion

28 10, in 1 - - - .

This article isprotected rights by All copyright. reserved. mechanism pro A inflammatory andnon res (LXA4 derivatives these of healing of maintenance whilelower, in stronger inflammatory and observed increased AD of skin affected In against of initiation and via induced likely highly pathways skin the of inflammation further response scratch induced AD of skin eicosanoid/ pro and status inflammatory 2 (Figure LA percentile reduced determined Linoleic acid deficiencywas described st inflammatory de PUFA allergen PUFA

reduced expression of the leukotriene receptors (BLT1 and CYS and (BLT1 receptors leukotriene the of expression reduced Accepted Article non in creased lig ii mdaos Fgr 2 (Figure mediators lipid olving - inflammatory , -

deficient diet is kn is diet deficient several

itch in

sensitized mice B

8, 10 8, pro ). We conclude th conclude We ). - ooaod levels docosanoid the patients

10 an increased ratio of pro ratioincreased an - . It seems that this altered itch altered this that seems It .

Pro . inflammatory enzyme pathways and all and pathways enzyme inflammatory conditions this skin disease n3 affected skin

atus srn ifamtr background inflammatory strong a

AD this /n6 - inflammatory signaling fetd kn f AD of skin affected - 53 eovn atvt was activity resolving Tee novel These .

-

28 MAR / PUFA and - hoi inflamma chronic , pro - patients

indicat various PUFAs ar own to induce AD induce to own a

was - further even resolvingenvironment presentin , systemic

metabolite

at i at the was 54

- n non in ) th conditions itch . es rsn in present

n AD n

even conditions in n3 n srn increased strong and

result present gnrl n ssei bcgon o AD of background systemic and general a

results / - any eitd i 5 via mediated mainly more a n6 eaoie ais n non in ratios metabolite - disorder - - A) fetd skin affected patients e patients inflammatory pro vs. to to be of relevance in a - ratios s - PUFA andseveraln3PUFA tion patients

higher increase of increase higher ee lo on in found also were in epidermal barrier defects barrier epidermal in e red

- n fetd skin affected in are like effects i effects like ugse t b mdae b 15 by mediated be to suggested AD 5 4, 8 5, 52 50, 48, 47, 45, unrbe kn to skin vulnerable

/ and and EPA and scratch signal scratch 5 50 35,

. ucedin affected skin 4 4, 49 45, 44, of of ven Compar eutn in resulting , is of is , displaying high cmaal t rtni dysregulat retinoid to comparable , a healthy looking skin looking healthy - Ti gnrl d general This . calculated metabolite/ n mice n motne o prevention for importance d o non to ed pro of ratio towards crucial .

-

, AD ing in non in ing resolving lipidresolvingmediators -

eue lvl o pro of levels Reduced pro /n6 AD O and LOX hc mgt indicate might which

o tendency no

- resolv no - 44 affecte patients - - - serum ad frhr inflammation further wards inflammatory patients(Table 1) importance for the progress the for importance PUFA - 46 sums of LOX and COX and LOX of sums L AA , aneac of maintenance T -

47, 48 47, while fetd skin affected R , bacterial in bacterial , - - -

si o AD of skin d 2) nlmaoy s pro vs. inflammatory metabolite ratios metabolite affected skin, which is which skin, affected metabolite leu confirmin 47, 48 47, . In ourstudy ation , seuain of ysregulation

mainly in AD in oree mediated kotriene is prone to a pro a to prone is f eovn and resolving of , here, we

- f the of eicosanoids

LOX - , 8 35 28, 7, patients ti pro this g transmitting fection ratios .

and - strategies

feedback resolving signaling - result inresult patients

chronic current further n3 . o in ion

were w e Itch

/ skin was and and ven n6

ere 51 - - - - - , .

This article isprotected rights by All copyright. reserved. 76 11, ( induce anti mainly thereby and signaling signaling cell inflammatory on activity inhibitory 75 mediate DP2 and DP1 via signaling response eico second A importantfor barrier formation ( metabolites, ALOX3 signaling nuclear are metabolites signaling inflammation pathways IFNγ either by induced is responseexpression inflammatory and homeostasis skin for relevance 12 controversial. increased of meaning biological The feedbackagainst th study our R increased and pro of formation the in resulting reviewedin vE Accepted Wh . Article

- .

affected skin affected

an l DP1 ile a d R d omn receptor, hormone

rvee in reviewed ,

present in atopicder inpresent following 69 61 in affected skin affected in but vD and - . 71

4 ) sanoid with a p a withsanoid bt partly but , 12 - 5 56 55, . o rslig n nrae lvl o pro of levels increased in resulting not aiu frs f 12/15 of forms Various derivatives

any mediates mainly Additional transmitting - HETE 12/15

of AD of

. ese

nuto o ltr pro later of induction he eety AO1B xrsin was expression ALOX12B Recently,

,

p 75

- pro O dfcec rsls n rtcin again protection in results deficiency LOX o - . h ma the patients

xilins

ly PGD2ly isalso known to be apotentendogenous ligandof PPAR γ e ocp ugss th suggests concept new A ( in the skin the in - also Ta inflammatory conditions

pain and itch response itch and pain ivotal role is PG is role ivotal which ble 2 ble matitis and psoriasisand matitis

and tri and

reviewedin act o mtblt oiiaig from originating metabolite jor - , hmtxs n p and chemotaxis resolving lipid me lipid resolving

especially in affected skin affected in especially ) s d pathways d . This reduced 12 reduced This .

a transmit can . s pro a as oxilins, were show were oxilins, A t A - LOX (15 endency for increased 15 increased for endency - eovn activities resolving - 42, 68

D2 LOX - LOX r kon n epesd n h si with skin the in expressed and known are - ,

reso

4 ). which may partly explain this pivotal role pivotal this explain partly may which

. It is described to induce pro induce to describedis It .

2, pathways

65 pro diators vn derivative lving ro in skin of 62, 63 62, - 59 67 - - LOX at - - nlmaoy inln, DP2 signaling, inflammatory ) . nlmaoy n hyperproliferative and inflammatory resolving

On the other hand, the ALOX12 andALOX12 thehand, other the On

. 12 . n pro r IL4 or

/ to be potent PPARα agonists and agonists PPARα potent be to 15LOX ratio indicates ratio 15LOX 55 - - - even , HETE 58 nlmaoy ligand inflammatory

fo

AD rvee in reviewed , the n PG and . n i or study our in und Indee - metabo (12/15

patients

AA 15 soitd ih retinoid with associated i

s - 72 HETE

- , a ligand of ligand a LOX metabolites were metabolites LOX d - 2 s mjr driver major a as D2 74 n further and - . in st lites LOX,

55, 56 55, PG

w non as legc airway allergic

D2 can initiate initiate can D2 s 42 . - - -

c as uch 60 inflammatory inflammator affected skin affected . confirmed in confirmed

) PPARδ a s

12

mediated biological a also may /15 remains induces 12

LXA4

- - 64 LOX LOX , a ,

4, y ,

This article isprotected rights by All copyright. reserved. DHA approach systemic a towards points ad In nutri topical or / and systemic for suitable not lipid t occurring endogenous Unfortunately, suggested maresins. and protectins lipoxins, resolvins, like n3 bioactive plus antagonists selective with receptors prostaglandin selective be treatmen compound multiple com single mainly addition In effects. detrimental as well as desired induce may pathway one pro in resulting PUFAs various eicosanoid Unfortunately v with preventive a how is question major The in affected skin ofAD PPAR reduced in γ and PPARα of expression reduced PPAR of concentration feedback inflammatory non the in while AD PPAR for 15 PGD2, of AD of skin affected in skin ofAD and4 PPAR endogenous potent as just in PPAR 16, 77, 78 77, 16,

Accepted Article- patient oe beneficial more pounds - nihd ih i i AD in oil fish enriched α/γ - iin or oe kolde about knowledge novel our dition, HDHA

ros prahs focus approaches arious v - arious eicosanoid arious - ) mediated signaling has been associated with anti with associated been has signaling mediated activation. . FABP . - s, patients targeting one pathwayonetargeting -

- originating 80 79, 76, 17, 11, KETE and 13 and KETE indicat s - - mediated signaling mediated

affec attracts ligands like n3 like ligands attracts . The. calculatedconcentrations sum ofthe of PPAR . ing In opposite, r opposite, In - - o a For patients. patients e si the skin ted .

- a In summary In

ligands precursors like HEPEs and HDHAs for pro for HDHAs and HEPEs like precursors – display - - - eaoim s eitd i te ae acd o ezms for enzymes of cascade same the via mediated is metabolism agonist eue ab reduced ligands derived KODE found in affected skin of AD of skin affected in found KODE hrpui apoc, w approach, therapeutic t -

- patients

strategy suggesting an antiansuggesting inflammatory and pro and inflammatory

ed o PPAR for s ing educed PPAR educed

tendencieseven or significantlevelsincreased inaffected are d15d12PGJ2, 12 d15d12PGJ2,

increased as well a well as , we observe we ,

characterizing chronic inflammatory conditions present conditions inflammatory chronic characterizing partly lt t dme ifamto ad kn healing skin and inflammation dampen to ility

- n genera on administered eitr are mediators 81 at distinct time points du points time distinct at -

PUFAs 11, 12 11, - as the and/ eitd transactivation mediated eosrtd y upeetto suis with studies supplementation by demonstrated

s PPAR s el as well . In our study in affected in study our In . PPAR tional a tional incr r hrpui srtg fr tpc derma atopic for strategy therapeutic or -

expression was expression and promot and - d inflammatory feedback mechanisminflammatoryfeedback srn anti strong l e

increased calculated sums and sums calculated increased ased γ - - KETE, - in current therap current in - target gene target metabolites resolving xrsin indicat expression s well as pharmaceutical applications. pharmaceutical as well s seiial sget blocki a suggest specifically e xrml lbl cmons n are and compounds labile extremely targeted

itch - inflammatory signalinginflammatory 15 e PPAR e - akr i non in markers - - KETE patients were even sufficient even were patients observed

a - expression levels expression ring allergic response may response allergic ring nlmaoy treatments inflammatory nti - oia co topical resolving - , 13 , - he mediated signaling mediated itch ies - n fetd kn but skin, affected in ligandswas increased

ed - skin, aoiis f these of majorities . We .

KODE, PGD2, PGJ2 PGD2, KODE,

rvnie topical preventive in affected skin of skin affected in

an 4, 6, 7, 52 7, 6, 4, the lipid - - suggest application fetd skin affected enabled sum of the the of sum - , result , mediators (reviewed . Blocking . sufficient .

Levels Levels th g of ng

3 14 13, anti 18, 19 18, at titis ing of a - , . ,

This article isprotected rights by All copyright. reserved. andwrote the manuscript. The authors declare no conflictof interest. RR interpretation, DT ASZ interpretation, biopsies, skin the organized and research clinical the coordinated JM and RL JG, immunohistochemistry, the performed UNKP the by supported also was and Sciences of Academy Hungarian the of scholarship research 2016 (DT) NN117020 and (ASZ) K128250 (RR), K109362 Funds and Development Research National Hungarian the by funded was project This Ackn LOX AD affected increased pro vs. inflammatory strongly In develops approach

Accepted Article- summary, patients is indicated in comparison to the non the comparison to inindicated is patients pathway andespecially LTB4 levels - - owledgement: 00005 and 00005 18

,

- erae n3 decreased

various Nw ainl xelne rga o te iity f ua Cpcte. CW Capacities. Human of Ministry the of Program Excellence National New 4 u prl as i non in also partly but pro o non to - inflammatory cascade are hallmarks of a chronic i chronic a of hallmarks are cascade inflammatory from limits the mind in keeping approache GINOP - fetd kn areas skin affected –

– planed and coordinated the study, analyzed the sampl the analyzed study, the coordinated and planed -

eovn lpd mediators lipid resolving /n6 aty rt te aucit n hle i dt aayi and analysis data in helped and manuscript the wrote partly - 2.3.2 - PUFA s

which - 15 - , n3 s, affected - 2016 can /n6 Currently . -

. 00050 grants 00050 also

- skin UA eaoie ratios metabolite PUFA

be ,

of tested under clinical conditions.

n srnl icesd itch increased strongly and - e of number low the ,

affected skin by additional by affectedskin

ua AD human ae on based . DT is a recipient of the of recipient a is DT . –

performed the PCR a PCR the performed and also by the by also and - patients hs knowledge these nflammation icesd ai o pro of ratio increased ,

. aie samples xamined h afce si of skin affected The – GINOP

es using LC using es - an epd n data in helped eitr and mediators present in the in present nalysis, MW nalysis,

, János Bolyai János increased 5 increased

Innovation u group our - 2.3.2 - - 15 , MS

– a - - - -

This article isprotected rights by All copyright. reserved. 11. 10. 9. 8. 7. 6. 5. 4. 3. 2. 1. References:

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- 8. ra N, Gelfand The EW. role leukotrieneof B(4) in allergicdiseases. Allergol

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- Martinz V, Krimmel C, Geisler A,Zhu H, al.Alterationset in k L,k Hanson LA, Kahu H,Dahlgren UI. Vitamin A deficiency a retinoid receptorX pathway. J Immunol 2002; bute to Inflammation and Impaired Late tis. Exp Dermatol 2011; 20:326

- 84. - 4.

- 30.

- 15.

- 6.

- This article isprotected rights by All copyright. reserved. 44. 43. 42. 41. 40. 39. 38. 37. 36. 35. 34.

Accepted Article scratching inspecial diet Fujii Nabe M, TomozawaT, J,Kohno S.Involvement of skin barrier dysfunction in itch deficiency disru Epp N,Furstenberger G, MullerdeK, JuanesS, Leitges Hausser M, I, al.et 12R 1841:390 Krieg FürstenbergerP, G. Therole of lipoxygenasesin epidermis. Biochim Biophys Acta 2014; 20:894 related behaviour in NC micewith atopic dermatitis Andoh Haza T, S, Saito A, Kuraishi Involvement Y. leukotrieneof B4 inspontaneous itch Pharm Bull 2010; 33:1050 in 5 Tsuji F,Aono H, Tsuboi T, Murakami T, Enomoto H, Mizutani K, al.et Role leukotrieneof B4 J Invest Dermatol 2007; 127:2042 ThromboxaneA2 induces itch Andoh Nishikawa T, Y, Yamaguchi andHT1 5 Kim DK, Kim HJ, Kim H,Koh JY,Kim KM, Noh etMS, al. Involvement of serotonin receptors 5 Pharmacol Sci2007; 105:41 5 Kuraishi Y, Ohtsuka NakanoE, T, Kawai S,Andoh NojimaT, H, al.et Possible involvement of ScientificWorldJournal 2007; 7:1307 N,Kim LusterAD. Regulation immune of cells by eicosanoid recept potential relevancefor skin disorders. Dermatology 2013; 225:304 signaling intheskin after systemic retinoid MihalyJ, Gericke J,Aydemir G,Weiss CarlsenK, H,Blomho stromal lymphopoietin. ProcNatl Acad SciS A U 2005; 102:14795 ablation inadult mouse keratinocytes generates an atopic dermatitis triggered by thymic M,Li Messaddeq N, Teletin M, Pasquali JL, Metzge - lipoxygenase metabolite in itch - lipoxygenase meta - 8. -

- 400. HT2 in12(S)

pts epidermalpts barrier function. J Cell Biol 2007; 177:173 - bolite HPETE - fed hairless mice. Eur J Pharmacol 2006; 530:152 - 3. - 7.

-

- - and allergy

associated responsesthrough TP receptors in theskin inmice. induced scratching in mice. Eur J Pharmacol 2008; 579:390 - - - associated response of mosquito allergy in mice. J 7. Miyamoto Nojima T, H,Narumiya S, K

- 28.

- induced itch - X receptorX ligand treatment in mice with r D, Chambon RetinoidP. receptor X - like skin lesion

- associated responses in mice.Biol ff R, al. et Reduced retinoid - 800. - ors. s. Exp Dermatol 2011; 11.

- uraishi Y. 82. - 6.

- lipoxygenase - related - - 4. -

This article isprotected rights by All copyright. reserved. 55. 54. 53. 52. 51. 50. 49. 48. 47. 46. 45.

Accepted Article Am JAm Respir Cell MolBiol 2008; 39:648 lacking 12/15 Anderss murine models colitis.of JImmunol 2013; 191:4288 mediator derived from omega Marcon R, Bento AF, Dutra RC, Bicca MA, Leite CalixtoDF, JB. Maresin 1,proresolving a lipid topical treatment infantileof eczema. Br J Dermatol 2012; 168:172 Wu SH, Chen XQ, Liu B, Wu HJ, Dong Efficacy L. and safety of 15(R/S) Pharmacol Physiol 2014; 27:242 Derived n MihalyJ, MarosvolgyiSzegedi T, A,Koroskenyi LucasK, Torocsik R, D, et al.Increased FADS2 Allergy Clin Immunol 1989; 83:450 prostaglandin E2 and leukotriene B4 arepresent inbiologically active concentrations. J Fogh Herlin K, T, Kragballe K. Eicosanoids inskin patientsof with atopic dermat dietary docosahexaenoicacid compositio RühlKoch R, C, Marosvolgyi T, MihalyJ, SchweigertFJ, Worm M, et al. Fatty acid dysregulation. Immunol Rev 2011; 242:233 Boguniewicz M, Leung DY. dermatitis:Atopic disease a of altered skinbarrierand immune dermatitis. JInvest De Schäfer L, Kragballe K. Abnormalities inepidermal lipid metabolism inpatients with atopic 2008; 100:66 transepidermal water loss in children withatopic dermatitis. Ann AllergyAsthma Immunol Yen CH, YS,Dai Yang YH, Wang Lee LC, JH, Chiang BL. Linol 14:460 skin inflammation caused by feedingspecial a diet to HR Fujii Tomozawa M, J, Mizutani N, NabeT, Danno K, Kohno S.Atopic dermatitis inflammation inspecial diet n of Fujii Naka M, - 6 polyunsaturated fatty acidsis mainlyresponsible for atopic dermatitis - 8. on CK,on Claesson HE, Rydell

- 6 PUFAs and Redu n serumof lipid inclasses micefollowing allergicsensitisation with or without shima H, TomozawaJ, Shimazaki Y, Ohyanagi C,Kawaguchi N,et al. Deficiency - - 73. lipoxygenase have attenuated airway allergicinflammation and remodeling.

rmatol 1991; 96:10 - fed hairless mice.Exp Dermatol 2013; 22:272 ced n - - enriched fish substitution.oil BrJNutr 2008; 99:1239 3 polyunsaturated3 fatty acids, exerts protectiveactions in - 8. - - -

3 3 inPUFAs Plasma Atopicof Dermatitis Patients. Skin Tormanen SwedmarkK, S, Hallgren A, Erjefalt JS. Mice 5.

- 56. - 5.

- 46.

- 98.

- 1 hairless1 mice.Exp Dermatol 2005; eic acideic metabolite levels and - 8. - methyl

- 7. -

- lipoxin A(4) in like pruritic skin - likepruritic itis: - 46.

- This article isprotected rights by All copyright. reserved. 65. 64. 63. 62. 61. 60. 59. 58. 57. 56.

Accepted Article mouseskin. PLoS ONE 2013; 8:e62643. mediated signaling involved GerickeJ, Ittensohn J, MihalyJ, Alvarez S,Alvarez R, Töröcsik D, et al.Regulation of retinoid and tetradecylthioacetic acid. J Invest Dermatol 2001; 116:702 Modulation keratinocyteof ge Westergaard M, Henningsen J,Svendsen ML, JohansenJensen C, UB, SchroderHD, etal. scratchings in mice. Eur J Pharmacol 2007; 554:30 Kim DK activation of TRPV1 and TRPA1receptors. Natl Proc Acad SciUSA 2012; 109:6721 12 Gregus AM, acid. IntJ Cosmet Sci 2014; 36:253 organoculture full of Gillbro JM, Al 400:378 production of PPAR Huang JS, JT,Welch Ricote BinderM, CJ, Willson TM, KellyC, et al.Interleukin psoriasisto vulgaris. EurJ Dermatol 2006; 16:141 lipoxygenase Setsu N, Matsuura H, HirakawaS, Arata J, Iwatsuki K. Interferon anti anddamage tissue in transgenicrabbit Serhan CN,Jain A, Marleau S,Clish C, Kantarci A,Behbehani B,etal. Reduced inflammation acute peritonitisproducing by proresolving mediators in mice. FasebJ 2011; 25:561 Yamada T, Tani Nakanishi Y, H,Taguchi R, Arita AraiM, H. secretory IgA levels. JAllergy Clin Immunol 2008; 122:633 Lipoxygenase deficiency protects micefrom allergic airways inflammation and increases AR,Hajek Lindley AR,Favoreto S,Jr., C - lipoxygenase - inflammatorylipid mediators. J Immunol 2003; 171:6856 , Kim HJ, Sung KS, Kim H,Cho SA, Kim KM, al.et 12(S) - 82.

Doolen S, Dumlao BuczynskiDS, MW, Takasusuki Fitzsimmons T, BL, al.Spinalet - - 2 expression in normal human epidermal kerati Bader WestmanT, M, Olsson MJ, Mavon A.Transcriptional changes in - derived hepoxilin A3 contributes to inflammatoryhyperalgesia via - gamma ligands inmacrophages12/15 by - thicknesshuman skin following topical application all of

in skin homeostasis by RAR and RXR agonists/antagonistsin neexpression and differentiation by PPAR - 61. arterR, SchleimerRP, Kuperman DA. 12/15

s overexpressing 15

- - 5. 3.

- Eosinophils promote resolution of 9 9 e3. - lipoxygenase and endogenous - HPETE induces itch nocytes andpathogenic a link - - lipoxygenase. Natu 65. - 12.

- gamma

- - induced 15 selective ligands - - trans retinoic 4 - - dependent associated re 1999;re - - 6. - - 8.

- This article isprotected rights by All copyright. reserved. 76. 75. 74. 73. 72. 71. 70. 69. 68. 67. 66.

Accepted Article eicosanoids regulategene expression through direct interactions with peroxisome KliewerSA, Sundseth SS, JonesSA, Brown PJ, Wisely GB, Koble CS, al.et Fatty acids and Med 2006; 12:148 Kostenis E, Ulven T. Emerging roles of DP and CRTH2in allergic infl Dermatol 2007; 16:331 cutaneous prostaglandin D production atopicon TakaokaA, Arai I, mechanical scratching inmice. EurJ Pharmacol 2005; 518:56 prostaglandin E2 acceleratethe recovery of cutaneous barrier disruption induced by Honma signal enhancement. Proc Natl Acad SciU SA 2013; 110:5205 inflammatory role of PGD2 in lung acute inflammation and therapeutic application its of Murata AritakeT, TsubosakaK, Y, Maruyama T, NakagawaT, Hori M, et al. Anti Allergy Clin Immunol 2010; isCRTH2 importantfor allergic skin inflammation after epicutaneous antigen challenge. J HeR, Oyoshi MK, Wang JY, Hodge JinMR, H,Geha RS. prostaglandinThe receptor D(2) 2005; 174:3703 prostaglandin D2 receptor DP2/CRTH2 increasesallergic Spik I,BrenuchonAngeli C, V, Staumont FleuryD, S, Capron M, et al.Activation of the 2006; 177:2621 essential rolein chronic allergicinflammation of the skin CRTH2 via receptor. JImmunol T,Satoh Moroi R, AritakeK, Urade KanaiY, Sumi Y, K, al. et Prostaglandin D2 plays a 2014; 1841:401 lipoxygenase Munoz PPARbeta/delta causespsoriasis a Romanowska M, Reilly L, Palmer CN,Gustafsson MC, FoersterJ. Activation of Jfactor. Invest Dermatol 2008; 128:110 enhances keratinocyte proliferation inpsoriasis and induces heparin Romanowska M, alYacoub N, Seidel H, Donandt S,Gerken H, Phillip S,et al. PPARdelta -

Garcia A, Tho Y, AraiY, I,Hashimoto Y, FutakiN, Sugimoto M, Tanaka etM, al. Prostaglandin and D2 - hepoxilin pathway in the mammalian epidermal barrier. Biochim Biophys Acta - - - 8. 9. 8. Sugimoto Futaki M, N,Sakurai Honma T, Y, et al.Role scratchof

- 58. mas CP, Keeney ZhengDS, BrashY, AR.importance The of the

- 9.

126:784 - likeskin disease in vivo. OnePLoS 2010; 5:e9701. - 90. -

24.

- likescratching behaviour in mice. Exp

inflammation in mouse. JImmunol - - 62. 10.

ammation. Trends Mol - binding EGF - - - like growth induced n

This article isprotected rights by All copyright. reserved. 81. 80. 79. 78. 77.

Accepted Article Dermatol 2008; 158:786 supplementation in atopic eczema: a randomized, double C,Koch Dolle S, Metzger M, RascheJungclas C, of PPARgamma by oxidized fatty acids. Nat Struct Mol Biol 2008; 15:924 T,Itoh Fairall L, Amin Inaba K, Szanto Y, A,Balint BL, et al.Structural basis for the activation activated receptor gamma. JBiol ketoneis core a moiety of natural ligands for covalent binding peroxisome to proliferator Shiraki Kamiya T, N, Shiki S, Kodama TS, KakizukaA, Jingami H. Alpha,beta 3:23 Dubrac S,Schmuth M. PPAR receptors in human skin. Horm Metab Res 2007; 39:96 Schmuth WatsonM, RE, Deplewski D, Dubrac S, ZouboulisGriffiths CC, Nuclear CE. hormone 23. proliferator

- 6.

- activated receptors alpha a - 92. -

alpha in cutaneous infl

Chem 2005; 280:14145 nd gamma. ProcNatl Acad Sci U SA 1997; 94:4318 H, Rühlet R, al. Docosahexaenoic acid (DHA) ammation. Dermatoendocrinol 2011;

- 105. - 53. - blind, controlled trial. Br J

- 31. - unsaturated

- - This article isprotected rights by All copyright. reserved. HXB3 HXA3 LXA4 LXB4 15 12 5 PGF2 8i TXB2 d15d12PGJ2 PGJ2 d15d12PGD2 PGE2 PGD2 LTC4 20 20 20 LTB4 20 15 12 11 8 5 AA 1: Table Tables: - - - - HETE HETE KETE ------

AcceptedPGF2 Article COOH COOH OH HETE HETE HETE HETE KETE KE * * * * *

α

* *

TE

-

LTB4 *

α

* *

- -

AA LTB4 * *

*

H 0.6 2.1 0.4 0.1 2.4 1.1 0.7 0.8 2.1 31.2 <0.1 0.3 1.1 6.8 4.7 0.3 0.8 0.9 0.1 3.3 0.8 1.6 42.5 1.0 0.7 6.1 314.8 - SE SE

(conc. in ng/ml) (conc. ± ± 0.5 ± 2.2 ± 0.1 ± <0.1 ± 2.1 ± 1.9 ± 0.5 ± 0.2 ± 2.4 ± 10.7 ± <0.1 ± 0.5 ± 0.3 ± 0.8 ± 2.2 ± 0.2 ± 0.5 ± 0.7 ± <0.1 ± 3.9 ± 0.4 ± 0.4 ± 11.1 ± 0.3 ± 0.3 ± 5.8 ± 74.8

0.5 0.2 0.5 2.1 2.4 99.6 1.0 0.4 0.5 2 D 1.0 1.4 0.1 0.2 0.1 3.3 1.0 0.9 2.6 52.5 0.1 0.4 0.6 6.2 6.8 0.2 2.6 65.6 - SE SE

(conc. in ng/ml) (conc. ± ± <0.1 ± 0.3 ± 0.6 ± 2.0 ± 2.1 ± ± 0.8 ± 0.5 ± 0.5 ± 168.4 ± ± 1.6 ± 0.8 ± <0.1 ± 0.1 ± ± 2.4 ± 0.8 ± 0.7 ± 2.5 ± 52.1 ± <0.1 ± 0.2 ± 0.6 ± 3.5 ± 2.9 ± 0.2 ± 2.6 92.2 < 0.1

0.37 0.43 0.29 0.33 0.57 0.35 1.00 0.51 0.17 0.67 Significance 0.74 0.64 < 0.40 0.13 0.28 0.65 0.75 0.81 0.53 0.38 0.76 0.25 0.77 0.37 0.48 0.29 0.01

This article isprotected rights by All copyright. reserved. half ng/ml). detectionof the limit (0.1 rep were ng/ml 0.2 of methodology used the of limit quantification the under were which (p<0.1). significance of tendency ( different significantly are letters bold AD conce The 13 13 9 LA PD1 MAR RvD2 RvD1 20 17 14 10 4 DHA PGE3 LTB5 18 15 12 8 5 EPA - - - - HEPE HEP HODE HDHA

Accepted- - - - - Article- - - - (D HDHA HDHA HDHA HDHA HEPE HEPE HEPE KODE HODE

*

* *

* -

E SE) (n=3) SE)

*

*

* *

ntration of lipid mediators in mediators lipid of ntration

* *

were measured by HPLC by measured were 3.0 81.7 19.2 5.1 4.3 365.2 0.3 0.4 2. 0.4 0.6 1.2 2.9 0.6 5.8 205 0.8 0.5 0.3 0.4 2.8 1.0 2

.2

Abbreviations see legend to figure 1. * For the marked substances values values substances marked the For * 1. figure to legend see Abbreviations ± ± 7.5 ± 1.1 ± 1.6 ± 89.5 ± 0.2 ± 0.1 ± 2.1 ± 0.5 ± 0.4 ± 0.9 ± 1.9 ± 0.4 ± 5.1 ± 79.9 ± 0.4 ± 0.3 ± 0.5 ± 0.2 ± 1.7 ± 0.4 ± 2.6 ± 39.6

P serum

< 0.05, Man 0.05, <

- MS

-

MS. Results in ng/ml are expressed as mean ± SD. Numbers in Numbers SD. ± mean as expressed are ng/ml in Results MS. samples from healthy volunteers (H volunteers healthy from samples 1.8 0.3 0.1 70.4 3.6 0.1 1.4 0.4 3.8 1.1 0.1 38.8 14.7 6.9 4.6 194.2 0.1 0.1 0.4 0.1 0.5 0.4 n Whitney test) and numbers in numbers and test) Whitney n

± ± 2.1 ± 0.1 ± 0.1 ± 30.0 ± 3.0 ± 0.1 ± 1.7 ± 0.3 ± 4.7 ± 1.4 ± 0.1 ± 35.8 ± ± 5.5 ± 0.9 ± 0.5 ± 173.4 ± 0.1 ± <0.1 ± <0.1 ± <0.1 ± 0.2 ± 0.5

0.53 0.29 0.12 0.0 0.18 0.05 0.37 0.75 0.74 0.89 0.13 0.24 0.45 0.04 0.78 0.43 0.12 < 0.20 0.31 0.66 0.27 0.01 3 -

SE) SE)

italic letters italic (n=3) laced with a value of of value a with laced and patients with with patients and

just identify a identify just This article isprotected rights by All copyright. reserved. EPA HXB3 HXA3 LXA4 LXB4 15 12 5 PGF2 8i TXB2 d15d12PGJ2 PGJ2 d15d12PGD2 PGE2 PGD2 LTC4 20 20 20 LTB4 20 15 12 11 8 5 AA 2: Table - - - - KETE HETE HETE ------

PGF2 KETE KETE COOH COOH OH Accepted ArticleHETE HETE HETE HETE

* * * * * α

-

*

LTB4 α

* *

*

* - -

AA LTB4

* * *

4.7 1.5 3.3 3.7 2.0 0.1 0.1 1.2 36.8 27.1 0.9 5.1 7.2 0.2 0.1 2.1 5.5 35.0 0.8 0.7 0.3 772.5 conc. in ng/g H 125.4 3.2 2.0 0.3 0.3 1.4 - SK

± 57.2± 4.3± 1.9± 0.1± 0.3± 1.2± 2.6± 1.4± 2.4± 3.2± 2.3± 0.1± 0.2± 0.5± 19.6± 14.7± 0.2± 5.9± 8.0± 0.1± 0.1± 0.8± 3.7± 33.3± 0.2± 0.3± 0.1± 83.0±

98.3 5.6 11.2 0.5 0.9 4.8 6.2 0.5 23.3 11.3 4.7 0.2 2.9 2.4 143.8 104.5 0.5 2.4 12.1 1.0 0.5 7.2 9.5 88.1 1.9 1.3 0.8 1563.2 N conc. in ng/g - SK

± 6.0± 0.8± 13.0± 4.9 ± 3.8± 0.2± 1.9 ± 0.3± 66.1± 102.0± 0.2 ± 2.2± 10.0± 1.4± 0.3± 7.3± 3.2± 60.8± 0.8 ± 0.4± 0.4 ± 353.4± ± 40.0± 4.0± 9.5± 0.5± 1.3± 4.2±

1.4 5.9 1.9 1.4 2.6 118.3 70.1 2.1 6.2 1.2 1408.0 conc. in ng/g A 97 10.5 9.1 0.7 0.1 25.8 10.5 0.8 8.6 6.9 5.7 0.1 2.1 2.7 66.1 62.3 0.5 - .8 SK

± 2.0± 4.1± 1.7± 0.6± 2.5± 143.3± 39.2± 0.7± 4.5± 0.1± 427.2± ± 21± 8.3± 9.4± 0.5± <0.1± 16.6 ± 9.4± 0.7± 2.2± 3.2± 8.2± 0.1± 0.3± 1.6± 12.5± 40.1± 0.5±

.8

0.50 0.54 0.39 0.10 0.29 0.23 0.26 0.07 0.11 0.08 0.02 H:N Significance 0.54 0.50 0.18 0.49 0.45 0.24 0.71 0.32 0.0 0.09 0.35 0.33 0.07 0.0 0.05 0.26 0.0 7 3 1

0.36 0.82 0.15 0.0 0.77 0.24 0.30 0.03 0.10 < 0.05 H:A 0.48 0.25 0.27 0.23 0.38 0.06 0.36 0.45 0.0 0.29 0.49 0.61 < 0.18 0.04 0.23 0.36 0.01 0.01 1 2

0.99 0.42 0.80 0.68 0.34 0.1 0.54 0.64 0.12 0.27 0.85 0.23 0.51 0.74 0.12 0.54 0.97 0.57 0.38 0.49 0.04 0.35 0.26 0.69 0.79 0.13 0.1 0.65 N:A 0 0

This article isprotected rights by All copyright. reserved. ng/g). the quantification limit usedof the methodology of 0.2 italic letters significancetendencyjust a identify (p<0.1). of expressed as bold mean in letters± Numbers SD. (n=3) The of concentration lipid media 13 13 9 LA PD1 MAR RvD2 RvD1 20 17 14 10 4 DHA PGE3 LTB5 18 15 12 8 5 - - - - HODE HDHA HEPE HEPE

------KODE AcceptedHODE ArticleHDHA HDHA HDHA HDHA HEPE HEPE HEPE *

* *

*

and affected and skin affected samples from patients with AD * *

*

*

* *

146.4 54.0 31.2 247.9 0.3 0.4 3.8 0.5 0.4 2.1 4.2 0.2 0.1 141.7 6.5 0.2 5.5 2.0 5.9 1.9 0.1

± 1.2± 4.5± 1.3± 0.1± ± 43.3± 19.6± 9.6± 36.4± 0.5± 0.4± 5.0± 0.5± 0.3± 1.3± 2.8± <0.1± 0.1± 37.3± 9.8± 0.4± 5.0±

tors in tors in

110.6 83.6 91.9 430.7 0.3 0.4 5.1 2.7 0.3 3.4 8.3 0.5 0.2 114.7 2.3 0.4 2.4 2.4 9.4 1.8 0.1

skin

± 64.3± 23.1± 72.0± 179.9± 0.2± 0.2± 4.0± 3.4± 0.3± 0.8± 7.5± 0.2± ± 29.5± 1.8± 0.6± 1.0± 1.0± 6.9± 2.4± <0.1± samples from healthy volunteerssamples (H from healthy

0.2

are significantly are ( different

0.2 0.7 33.9 9.4 1.1 0.4 153.7 16.5 0.7 2.7 10.0 5.8 5.1 0.3 377.7 211.0 59.7 311.4 0.7 0.3 1.5 ng/g were with halfng/g valuea replaced detectionof of the limit (0.1

(A

- SK) * valuesFor marked selectedthe substances were under which ± 0.1± 0.6± 27.1± 6.7± 0.6± 0.1± 46.0± 15.4± 0.6± 4.5± 7.1± 0.6± 4.3± 0.2± ± 190.0± 135.9± 37.3± 46.0± 0.2± 0.3± 0.5±

(n=3)

were measured by HPLC by were measured

P

< 0.05, Mann Whitney and< Mann 0.05, test) in numbers 0.34 0.92 0.24 0.43 0.14 0.57 0.38 0.51 0.77 0.36 0.64 0.50 0.96 0.20 0.47 0.17 0.22 0.16 0.81 0.99 0.74 - SK) SK) (n=3), non

- MS - affected skin samples 0.37 0.3 0.11 0.28 0.03 < 0.74 0.40 0.33 0.51 0.13 0.96 0.28 0.12 0.11 0.12 0.27 0.13 0.29 0.93 0.48 - 0.01 MS. ResultsMS. in are ng/ml 9

0.08 0.18 0.53 0.33 0.04 0.90 0.20 0.29 0.34 0.12 0.86 0.13 0.11 0.28 0.19 0.53 0.93 0.1 0.41 0.31 0.18 4 (N

- SK)

This article isprotected rights by All copyright. reserved. HXB3 LXB4 acid, eicosatetraenoic eicosatetraenoic TXB2 D2, 15 F2 prostaglandin PGD2 H2, prostaglandin 20 LTB4 20 acid, HETE - 13 acid, octadecadienoic RvD1 D1, protectin hy 14 acid, docosahexaenoic 4 B5, leukotriene 18 acid, eicosapentaenoic 12 acid, 5 gamma DHA acid, docosapentaenoic blue. ALA with marked Abbreviations: are downregulated 20% than more are which derivatives and o serum MS HPLC our 1: Figure Figure legends:

- 8 HEPE HEPE droxy Accepted- - - Article - deoxy COOH oxo hydroxy - -

- -

20 -

hepoxilin B3, MARhepoxilin 9,11 - linolenic acid, DHGLA acid, linolenic - ptet wt A i cmaio t haty otos r mre wt red with marked are controls healthy to comparison in AD with patients f docosahexaenoic acid, 20 acid, docosahexaenoic - 12 HETE - - -

- Δ HEPE HEPE AA

hydroxy - 5 12,14 -

ceai darm f eiaie wih ee any eemnd using determined mainly were which derivatives of diagram Schematic - - - hydroxy hobxn B, 8i B2, thromboxane eicosatetraenoic acid, 11 acid, eicosatetraenoic octadeca hydroxy - MS competence. Derivatives which are more than 20% upregulated in upregulated 20% than more are which Derivatives competence. MS -

– 20

α

- acid, - - rsalni J, d15d12PGD2 J2, prostaglandin 20 , PGJ2 PGJ2 , HDHA - - leukotriene B4, 20 B4, leukotriene carbohydroxy - - - 12 - ioaereoc cd 15 acid, eicosatetraenoic - hydroxy

ioaeteoc cd 8 acid, eicosapentaenoic dienoic acid,dienoic

13 resolvin D1, RvD2 RvD2 D1, resolvin 12 - alpha hydroxy - - - - HODE HODE -

- KETE HDHA HDHA - 4

prostaglandin J2, PGE3 PGE3 J2, prostaglandin

maresin. - - prostaglandin D2, PGE2 PGE2 D2, prostaglandin hydroxy - HEPE HEPE - - ioei ai, EPA acid, linolenic eicos - lipoxin B4, LXA4 LXA4 B4, lipoxin - -

dihydro - - - - ioaeteoc aci eicosapentaenoic -

PGF2α LA acid, docosahexaenoic

13 - rcioi ai, LTC4 acid, arachidonic 12 14 5 atetraenoic acid, LTB4 LTB4 acid, atetraenoic HDHA HDHA - -

- - HETE

- - - hydroxy docosahexaenoic acid, 10 acid, docosahexaenoic - oxo hydroxy COOH HETE 18 - gamma - - -

- - ioaereoc acid eicosatetraenoic hydroxy

8 -

eovn 2 9 D2, resolvin

20 - - 5 – iso LTB4 LTB4 - - -

9,11 - hydroxy docosahexaenoic acid, 17 acid, docosahexaenoic 11 - hydroxy - - - HETE HEPE HEPE rsalni Fα 5 F2α, prostaglandin linolenic acid, AA acid, linolenic - -

hydroxy - - - - lipoxin A4, HXA3 HXA3 A4, lipoxin eicosapentaenoic acid, LTB5 LTB5 acid, eicosapentaenoic - octadecadienoic acid, 13 acid, octadecadienoic -

20 prostaglandin E3, d15d12PGJ2 E3, prostaglandin ioaeteoc cd DPA acid, eicosapentaenoic 15 - - , 15 d, - - - - eicosatetraenoic eicosatetraenoic acid, 8

carbohydroxy

docosahexaenoic acid, PD1 PD1 acid, docosahexaenoic - 15 deoxy rsalni E, PGF2 E2, prostaglandin 8 - - - HODE HODE - eicosatetraenoic acid, 12 acid, eicosatetraenoic hydroxy - -

hydroxy ektin C, PGH2 C4, leukotriene ektin B, 20 B4, leukotriene - - HEPE HEPE

- - Δ 15 , linoleic acid, GLA GLA acid, linoleic HDHA 12,14 - -

-

9 eicosapentaenoic - - arachidonic acid, arachidonic eicosatetraenoic KETE - - - - hydroxy - leukotriene B4, leukotriene KETE

- - prostaglandin eoii A3, hepoxilin

- 15 10 HDHA HDHA - - -

hydroxy hydroxy - - KODE KODE 15

- 5 10,12 - HETE HETE - - - - oxo oxo

OH α 17

– ------

This article isprotected rights by All copyright. reserved. controls(H SK) proteins) binding acid fatty and genes (5 pathways metabolic levels 3: Figure abbreviations see legend 12 (H controls affected PPAR of metabolites 15LOX / 12LOX summarised affected skin (N (12 mediators (N skin PUFA (H volunteers healthy (H controlshealthy (A affected in LA and MAR PD1, RvD2, RvD1, 5 and TXB2 d15d12PGD2, PGF2, PGE2, PGD2, LTC4, PUFA summarised HODEs, HDHAs, HEPEs, calculated were Ratios HODEs. HDHAs, HEPEs, HETEs, summarized 5 from and LA DHA, EPA, AA, from originating metabolites from calculated are Sums LA). DHA, EPA, (AA, 2 Figure

Accepted- Article LOX (n=6) - eaoie wr dslyd s ecnie mut fr fetd (A affected for amounts percentile as displayed were metabolites f aiu ezms n receptors and enzymes various of -

K o AD of SK) – - ligands (A

: 12 - Calculated upregulation and downregulation of the n the of downregulation and upregulation Calculated - A acltd us n rto o aiu e various of ratios and sums Calculated (A) - n non and SK) K. brvain: 5 Abbreviations: SK). K, non SK), - lipoxygenase, 15 lipoxygenase, - - HETE, LTB4, TXB2, PGE2, PGF2) PGE2, TXB2, LTB4, HETE, LOX, 12 LOX, - (n=6) SK) ofAD ( 1d2G2 12 d15d12PGJ2, - - ains oprd o kn f elh cnrl (H controls healthy of skin to compared patients SK) as well as serum of AD serumof asas well SK) - fetd kn (N skin affected . - -

fetd kn (N skin affected SE). (B) SE). - - - K, non SK), ) O, 12/15 LOX, LOX, 15 LOX, of lipid mediators (pro mediators lipid - patientscompared to skin ofhealthy controls(H figure 1. - LOX The c The - - LOX, COX and 8 and COX LOX, fetd kn (N skin affected - - LOX KETE, - –

O, O ptwy, PR eetr ad PPAR and receptors PPAR pathways, COX LOX,

alculated amounts of AA of amounts alculated

- 15 SK) involved in PUFA in involved – - K o AD of SK)

- like listed in table 1 and 2 and 1 table in listed like lipoxy 5 15 - lsee ad acltd o dfeet functio different for calculated and clustered (n=6) - ioyeae 8 lipoxygenase, metabolites as well as pro as well as metabolites - KE - inf / pro / inf - genase, COX genase, patients(D E 13 TE,

in skin biops skin in - - hydroxylation (8 hydroxylation f AD of K o AD of SK) - ains oprd o kn f healthy of skin to compared patients - KETE - - OE PD, G2 n 4 and PGJ2 PGD2, KODE, res) originating from from originating res) - csnis docosano icosanoids,

- metabolite signaling metabolite patients in comparison to to comparison in patients - ) SE) in comparison to serumfromcomparisonin to SE) - OH and pro and – - ies from affected (A affected from ies ains oprd o kn of skin to compared patients -

, DHA , cyclooxygenase. ormalized mRNA expression mRNA ormalized –

- 8 OH) pathways as well as as well as pathways OH) - - yrxlto pathway, hydroxylation and (E) calulated sums calulated (E) and - - resolving , EPA , SK). - rm P from inflammatory -

K, non SK), - - C Sm f itch of Sum (C) SK). (D) R

and LA and d ad PUFAs and ids in affected (A affected in AA, EPA, DHA EPA, AA, Fs HETEs, UFAs, ( LXA4, LXB4, LXA4, Other listed Other - - SK), non SK), HDHA) - - affected derived healthy - ( atio of of atio target LTB4, nal nal

in - - - This article isprotected rights by All copyright. reserved. Accepted Article the markedby the green arrow. in proteins both of presence strong while arrows, black (A patients (H healthy in staining ALOX12B and COX1 of images immunohistochemical Representative dermatitis. atopic with 4 Figure : - Expression of COX1 and ALOX12B in the skin from healthy controls and patients and controls healthy from skin the in ALOX12B and COX1 of Expression SK). Strong presence of presence Strong SK). - SK), non affected skin of A of skin affected non SK), Bars, 10 ALOX12B and COX1 proteins in epidermis is marked by marked is epidermis in proteins COX1 and ALOX12B 0

μm.

D - patients (N patients

- SK) and affected skin of AD of skin affected and SK) dermal nitaig el are cells infiltrating - This article isprotected rights by All copyright. reserved. Accepted Article

This article isprotected rights by All copyright. reserved. Accepted Article

This article isprotected rights by All copyright. reserved. Accepted Article

This article isprotected rights by All copyright. reserved. Accepted Article