allergen skin challenges.pdf

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Clinical StudyCirculating Conventional and Plasmacytoid Dendritic CellSubsets Display Distinct Kinetics duringIn VivoRepeated

Allergen Skin Challenges in Atopic Subjects

Stelios Vittorakis,1,2 Konstantinos Samitas,1,2 Sofia Tousa,2

Eleftherios Zervas,1 Maria Aggelakopoulou,2 Maria Semitekolou,2

Vily Panoutsakopoulou,2 Georgina Xanthou,2 and Mina Gaga1

th Respiratory Department and Asthma Centre, Athens Chest Hospital, Athens , Greece Cellular Immunology Laboratory, Division o Cell Biology, Center or Basic Research,

Foundation or Biomedical Research o the Academy o Athens, Athens , Greece

Correspondence should be addressed to Konstantinos Samitas; [email protected]

Received February ; Accepted April ; Published April

Academic Editor: Petros Bakakos

Copyright Stelios Vittorakis et al. Tis is an open access article distributed under the Creative Commons AttributionLicense, which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properlycited.

Upon allergen challenge, DC subsets are recruited to target sites under the inuence o chemotactic agents; however, detailspertinent to their trafficking remain largely unknown. We investigated the kinetic proles o blood and skin-inltrating DC subsetsin twelve atopic subjects receiving six weekly intradermal allergen and diluent injections. Te role o activin-A, a cytokine inducedin allergic and tissue repair processes, on the chemotactic proles o DC subsets was also examined. Plasmacytoid (pDCs) andconventional DCs (cDCs) were evaluated at various time-points in the blood and skin.In situactivin-A expression was assessed inthe skin and its effects on chemokine receptor expression o isolated cDCs were investigated. Blood pDCs were reduced h aferchallenge, while cDCs decreased gradually within h. Skin cDCs increased signicantly h afer the rst challenge, inverselycorrelating with blood cDCs. Activin-A in the skin increased h afer the rst allergen challenge and correlated with inltratingcDCs. Activin-A increased the CCR/CCR expression ratio in cultured human cDCs. DC subsets demonstrate distinct kineticproles in the blood and skin especially during acute allergic inammation, pointing to disparate roles depending on each phaseo the inammatory response. Te effects o activin-A on modulating the chemotactic prole o cDCs suggest it may be a plausibletherapeutic target or allergic diseases.

1. Introduction

Allergic diseases, such as asthma and atopic dermatitis, areon the rise in western societies and pose a signicant burdenor patients and health care systems. Allergic inammationrelates to excessive helper type (H2) cell-mediatedresponses against innocuous environmental allergens. Den-dritic cells (DCs) are known sentinels o the immune sys-tem entrusted with the chie task o antigen recognition,presentation, and cell activation. Different DC subsetswith diverse unctionalities have been described, dependingon the level o expression o specic surace markers, theiractivation status, and anatomic location. Tere are two major

human DC subsets: myeloid or conventional DCs (cDCs),generally considered immunogenic [,], and plasmacytoidDCs (pDCs), which can exhibit suppressive unctions onallergen-driven H2cell-mediated responses []. Pertinent tothe skin, cDCs constitute the major resident DC populationin normal human dermis and are characterized by CDcexpression (also known as blood dendritic cell antigen-(BDCA)-) [,]. Plasmacytoid DCs are present in the skinand can be readily identied by expression o CD, alsoknown as BDCA- [,].

Increased numbers o DC subsets have been previouslyreported in the blood, nasal, and/or lung mucosa o sub-

jects with atopy, allergic rhinitis, and/or asthma [],

Hindawi Publishing CorporationBioMed Research InternationalVolume 2014, Article ID 231036, 14 pageshttp://dx.doi.org/10.1155/2014/231036
http://dx.doi.org/10.1155/2014/231036http://dx.doi.org/10.1155/2014/231036


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BioMed Research International

suggesting that specic DC subgroups are induced inresponse to allergic inammation. Nevertheless, ew clinicalstudies have addressed DC kinetics in allergic responses,and these involve mostly allergen inhalation or segmentalbronchial allergen challenge []. Understanding o thetrafficking o human DCs upon allergen challenge in vivois

essential or controlling the balance between immunity andtolerance.DC migration is guided by the rapid upregulation o

adhesion molecules and chemokine receptors in responseto chemokine and cytokine gradients generated by tissue-resident and inltrating immune cells. Activin-A is apleiotropic cytokine-member o the GF- superamily oproteins. It is produced by inammatory and structuraltissue cells and acts as an important regulator o allergicinammation [] and skin repair processes []. Inact, recent studies have revealed potent anti-inammatoryeffects o activin-A in vivoduring allergen-induced cutaneoussensitization []. Activin-A is produced by different DCsubsets, promotes DC differentiation, and affects the abilityo mature DCs to take up antigens []. In addition, activin-A is involved in the differentiation and migration o humanLangerhans dendritic cells, mostly through the regulation ochemokines and chemokine receptor networks [,]. Still,the effects o activin-A on shaping the chemotactic responseso human DC subsets during exposure to allergen in vitroremain elusive.

In the present study, we hypothesized that circulatingcDCs and pDCs exhibit different kinetic proles in vivo uponrepeated skin allergen challenges, reecting their distinctroles during acute and established chronic inammation.o explore this, we used a well-established human in vivomodel o repeated skin allergen challenges. Furthermore,we examined activin-A expression in the inamed skin andexplored possible correlations with DC subset inltrationollowing allergen challenge. Finally, the effects o activin-A on the chemokine receptor prole o allergen stimulatedhuman CDc+ DCs were also investigated.

2. Methods

.. Study Population and Design. Healthy nonsmoking vol-unteers between the ages o and were initially screenedand subjected to skin prick testing (SP) to a panel o common aeroallergens (HAL Allergy, Benelux). otal blood

counts, serum biochemistry, total IgE levels measurements,and spirometry were perormed. A total o twelve subjectswith strong positive SP reaction to Dermatophagoides

pteronyssinus, the European house dust mite, were enrolledin the study. Subject characteristics are summarized inable . All subjects signed an inormed consent orm beoreenrolment. Te study was approved by the Sotiria HospitalResearch Ethics Committee and the Greek National Orga-nization or Medicines and was conducted according to theDeclaration o Helsinki principles.

All participants were ree o atopic symptoms or at leastone month beore and during the study. Participants hadno history o inection and had not received any treatment

30 BU intradermal allergen with equal volume diluent controlSkin measurements perormed at 15 min and1 hour

Days: 30 10 2 8 15 22 29 36 37

Screening period

Allergen and diluent skin biopsy

K3EDTA collected whole blood sampleFACS-DC acquisition

F : Study design. Flow diagram showing the time-pointswhen the skin challenges with allergen and diluent were perormedand when the samples were taken. Te dose oDermatophagoidespteronyssinusadministered was BU at the allergen site with equalvolume o diluent at the opposing site every week (solid arrows).A screening period o weeks was introduced to clinically veriythat subjects did not exhibit seasonal allergic symptoms or anupper/lower respiratory inection.

with oral/inhaled corticosteroids, antihistamines, anti-IgE,or antileukotrienes or one month beore and during thestudy. Each subject received a total o six weekly intradermalinjections o allergen, to which they were sensitive, anddiluent on the extensor aspect o the lef and right orearms,respectively. Skin biopsies were obtained h afer the rstchallenge and h afer the last challenge at both allergenand diluent sites. Whole blood was taken beore the rstchallenge, h afer the rst challenge, and just beore eachbiopsy acquisition. Te study design is depicted inFigure .

.. In Vivo Allergen Challenges. A -gauge needle wasused to deliver L ( BU) o allergen aqueous solution

(Dermatophagoides pteronyssinus, Allergopharma JoachimGanzer KG, Germany) intradermally at the same site onthe extensor aspect o the lef orearm. Te same diluent

volume (.% sodium chloride) was administered in thesame manner on the extensor aspect o the right orearm.In this way, each patient served as his/her own control.Te challenge tests were perormed at the same time eachday. Measurements o skin reactions were perormed at min, h, and h, as previously described [], by a singleinvestigator throughout the study.

.. Preparation o Skin Biopsies. Skin specimens wereobtained rom the centre o both allergen and diluent sites

in each subject using a mm punch biopsy tool (StieelLaboratories). Local anaesthesia was induced by injectingsubcutaneously . mL o % lidocaine hydrochloride. issuesamples rom the same arm afer the last challenge weretaken . cm apart. Afer appropriate orientation and han-dling, specimens were cut in hal and one o the resultingtissue samples was embedded in OC medium and snap-rozen in isopentane (BDH Chemicals) precooled in liquidnitrogen, while the other was xed in ormalin overnight andembedded in paraffin. Frozen specimens were stored at C.

.. Immunohistochemistry. Paraffin sections - mmthick were deparaffinised in xylol, rehydrated in graded


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T:SubjectcharacteristicsandsummaryofresultsforperipheralandtissueD

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alcohol series, and immunostained or activin-A, aspreviously described []. In brie, the alkaline phosphatase-antialkaline phosphatase method was used, and specicantibody binding was visualized using Vectastain ABC-APkits and the Fast-Red chromogen (rabbit anti-goat IgG, AK- Vector Laboratories). All incubations were perormed at

room temperature. Washes were perormed in PBS. Normalrabbit serum (%) was used to reduce nonspecic binding.An affinity-puried polyclonal goat antibody against humanactivin-A and a goat IgG control antibody (which served asnegative control) were used as primary antibodies (AFand AB--C, resp., R&D Systems).

.. Immunouorescence Staining. o determine the numberso cDCs andpDCs in the skin, we applied an immunostainingtechnique to serial cryosections ( m). Slides were warmedup at room temperature (R) or min, xed in ice cold(C) acetone or min, air dried or min, and washed inPBS. Sections were then blocked with normal goat serum (S-

, Vector Laboratories) and incubated overnight at

Cwith unconjugated mouse anti-human CDc (BDCA-) orthe determination o cDCs or mouse anti-human CD(BDCA-) or pDCs (-- and --, resp.,Miltenyi Biotec). A secondary goat anti-mouse antibody con-

jugated with AlexaFluor or goat anti-mouse conjugatedwith AlexaFluor (A and A, resp., MolecularProbes) was applied or min at R. Negative controlswere obtained by substitution o the primary antibody withthe same concentration o the corresponding IgG isotypecontrol (MAB and MAB, resp., R&D Systems). Slideswere rinsed and then counterstained with Hoechst (H,Molecular Probes).

.. Conocal Laser Microscopy. Skin sections processed withimmunouorescence staining were examined using a cono-cal laser microscope. Fluorescent imageso the tissue sectionswere acquired, using a Leica DMI inverted microscopewith DIC optics (Leica CS SP). Hoechst was excited bythe nm laser diode and the uorescence was collectedusing a long-pass (LP) emission lter; the green (AF)antibody was excited by the nm argon ion laser line andthe uorescence was collected using a band-pass (BP) emission lter; the red (AF) antibody was excited bythe nm green helium-neon laser line andthe uorescencewas collected using an LP emission lter. issue sections

were visualised using the conocal microscope at , ,-pixel resolution through a HCX PL APO CS . .DRY UV with eight-times averaging in sequential scanning(multitrack) mode with the pinhole set to obtain an opticalsection o approximately mm in all channels.

.. Quantication o DCs and Activin-A Positive Cells.Images o tissue sections were recorded using a computerizedimage analysis system (AxioVision, Carl Zeiss). Cells stainedor activin-A were counted in the epidermis and dermis byusing an AxioScopeA light microscope (Carl Zeiss). Resultswere normalized to the area o the epithelium and to thelength o the basement membrane and expressed as number

o cells per mm2. Quantitative measurements o BDCA-and BDCA- positive cells in skin tissue specimens wereperormed as previously described [,]. DCs that showedpositive staining were counted to a dermis depth o m.Stained DCs were regarded positive when showing DC

morphology and were expressed as number o cells per mm2

o dermis area. Activin-A, BDCA-, and BDCA- stainingwas perormed in serial tissue sections, which were codedand examined in a blinded manner at the end o the studyby two investigators. Te intra- and interobserver variationswere calculated to be less than % and %, respectively.

.. CDc+ DC Isolation and In-Vitro Stimulation. Peripheralblood mononuclear cells (PBMCs) were obtained by Ficollgradient centriugation and CDc+ DCs were isolated usingthe CDc+ (BDCA-+) human dendritic cell isolation kit,(Miltenyi Biotec --). Briey, in a rst step, PBMCswere magnetically labeled with CD microbeads. Using

this approach, CD+

cells were depleted by separation overa MACS column which was placed in the magnetic eldo a MACS separator. In a second step, CDc+ DCs, inthe B cell-depleted ow-through raction, were magneticallylabeled with CDc-biotin and antibiotin microbeads. Uponseparation, the labeled CDc+ DCs were retained withinthe column and eluted afer removing the column rom themagnetic eld. Tis two-step procedure allows the isolationo puried CDc+ DCs (with a purity>%). A total o

4 CDc+ DCs were placed in at-bottom plates andcultured or h with (a) medium, (b) ng/mL LPS, or (c) g/mL Dermatophagoides pteronyssinusin the presence orabsence o ng/mL recombinant activin-A (R&D). LPS is a

LR ligand extensively used or the activation o DCs andserved as a positive control []. Following h o culture,cells were stained with uorochrome-conjugated antibod-ies against human CCR (CD), CCR (CD), CCR(CD), CXCR (CD), and CCR (all rom Biolegend)and analyzed by ow cytometry.

.. Flow-Cytometric Analysis. Peripheral blood was drawnand processed or ow-cytometric analysis within mino collection. Te identication o peripheral DC subsetswas based on two different three-color assays (IOest PN-A myeloid subset and PN-A plasmacytoid sub-

set, Immunotech, Marseille, France) and was perormed aspreviously described []. Whole blood ( L) was mixedwith L o each monoclonal antibody or appropriatelabelled isotype control cocktail, vortexed or sec, andincubated or min at R, protected rom light. Te sampleswere lysed and xed (VersaLyse PN-IM and xativesolution PN-IM, Beckman Coulter, Kreeld, Germany),washed, resuspended in PBS, and kept on ice until owcytometric analysis. At least events were acquired ona dual-laser Beckman-Coulter FC. Further analysis o theow-cytometric raw data was perormed with the FLOWJOsofware (ree Star Inc., USA). Te gating strategy used orthe analysis o DC subsets is provided in Figure .


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Early-phase reaction

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F : Early- andlate-phase reactions. Mean diameter o skin reactions afer each allergen challengewith Dermatophagoides pteronyssinusmeasured at min and h (a), as well as h (b), is presented in mm. No statistical signicant differences were observed between challenges

or at different time-points. Values are expressed as mmSEM.

.. Statistical Analysis. Data are expressed as median withinterquartile range (rst and third quartiles) unless speciedotherwise. Normality was assessed using D AgostinoPearson omnibus normality tests. Data differed rom normaldistribution and were, thus, analyzed with non-parametricstatistics. Statistical analysis o skin reactions measured at min, h and h afer each challenge was perormed bytwo-way ANOVA mixed model, ollowed by Bonerroniposthocanalysis. DC kinetics in peripheral blood as well as theanalysis o the number o tissue DC subsets at different time-

points were perormed by repeated measures nonparametricone-way ANOVA (Friedman test), ollowed by Dunns posthocanalysis or all pairs. Te correlation between DC subsetsin skin tissue and peripheral blood at different time-points, aswell as between activin-A tissue expression and DC subsetsin the skin, was perormed by nonparametric Spearmancorrelation tests. wo-group analysis o the effects o activin-As on chemokine receptor expression by allergen-stimulatedCDc+ DCs was perormed by the Mann-Whitney test.A value o. was considered statistically signicant.Data storage and analysis were perormed with statisti-cal analysis sofware (GraphPad Prism v, GraphPad Inc.,CA, USA).

3. Results

.. Early- and Late-Phase Allergen-Induced Cutaneous Reac-tions. Te mean diameter o skin reactions afer each aller-gen challenge measured at min, h, and h (Figure ).Statistical analysis showed no diameter differences betweenchallenges or at different time-points, minimizing the possi-bility o induction o tolerance during the in vivo protocol.No indurations were observed at the diluent site. No diluentand/or allergen-related adverse events were observed in anyo the subjects.

.. DC Subsets Exhibit Different Kinetic Patterns in thePeripheral Blood Following Repeated Allergen Skin ChallengesIn Vivo. Circulating cDCs and pDCs were assessed beoreallergen challenge, h and h afer the rst allergen chal-lenge,and h afer the sixth challenge(Figures(a) and (b),resp.). Interestingly, our data revealed signicant differencesor both cDC and pDC numbers in the peripheral blood oatopics at distinct time-points ollowingin vivoallergen skinchallenge, compared to baseline ( < 0.05, resp.). Post hocanalysis revealed that changes in the pDC population were

very rapid, as they were signicantly decreased h afer therst allergen challenge and returned to baseline afer h(Figure (c)). On the other hand, cDCs started to decreaseby the rst hour afer challenge and continued to decrease,reaching signicantly lower levels at h, with a later-ontrend to return to baseline afer weeks (Figure (d)).

.. Conventional DCs Are Recruited to the Skin Early Follow-ing In Vivo Allergen Challenge. DC subsets were examinedin the skin at the allergen and diluent sites h afer the rstandsixth challenges, time-points at which DC inltration hasbeen reported to peak []. Our ndings demonstrated that

pDCs and cDCs were mostly located in the subepitheliumwithin m range rom the epidermis but occasionallyalso extended deeper into the dermis (Figures(a)(c)and(a)(c), resp.). Pertinent to pDC recruitment, there was atrend or increased pDC numbers at the allergen site afer thenal allergen challenge (Figure (d)). In contrast, one-wayANOVA revealed signicant differences between the distincttime-points or cDCs ( = 0.0015), with post hoc analysisdemonstrating a signicant increase at the allergen site hafer the rst allergen skin challenge, compared to the diluentsite ( < 0.05,Figure (d)). Although a trend or increasedcDCs was also observed at theallergen site h afer the sixthchallenge compared to the diluent site, this difference was


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Gate1 Gate2 Gate3

45.9% 3.41%

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(d)

F : DC subsets exhibit different kinetic patterns in the peripheral blood ollowing repeated allergen skin challengesin vivo. Gatingstrategies utilized to identiy cDC and pDC subsets in the peripheral blood by ow cytometry. (a) For cDC identication, a -step analysiswas perormed. Initially, CD33pos cells were selected (Gate ) to differentiate between mature lymphoid cells or lymphoid precursors

(CD33neg) rom other cells o myeloid origin that include cDCs. Next, all CD (14 + 16)dim to neg/IL3pos cells are selected in Gate to

exclude monocytes, macrophages, NK cells, and neutrophils. Gate is drawn around CD33bright/IL3pos cells, so cDCs are characterized

asCD33bright/IL3pos/CD(14 + 16)dimtoneg. (b) Regarding pDCs, initially all CD123pos cells are selected (Gate ) and then gated onthe basis o

CD(14 + 16)neg expression (Gate ) to exclude monocytes, lymphocytes,and most granulocytes. Gate is drawn around CD123bright/IL3bright

cells, strictly selecting pDCs and excluding basophils, so pDCs are characterized as CD123bright/IL3bright/CD(14 + 16)neg. RepresentativeFACS plots are shown. Te percentages o pDCs (c) andcDCs (d) in the peripheral blood at baseline and ollowing in vivo allergen challengesare shown. Data are expressed as median with interquartile range (rst and third quartiles).


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F : PlasmacytoidDC kinetics in theskin upon in vivoallergen challenge. Immunouorescence staining was perormed on skin biopsiesand examined by conocal microscopy. Counterstaining was perormed with Hoechst to visualize nuclear DNA (blue, column ). PDCs werestained with a monoclonal antibody against BDCA- and envisioned with a secondary goat anti-mouse antibody conjugated with AF(green, column ). Column is the result o merging columns and . Representative microphotographs () o pDCs are shown hafer the rst challenge at the diluent (a) and allergen sites (b) and afer the sixth challenge at the allergen site (c). PDC numbers were notsignicantly altered between different time-points, although a trend or increased pDCs was observed afer the sixth allergen challenge (d).Data are expressed as median with interquartile range (rst and third quartiles). < 0.05, WBC: whole blood cells.


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F : Conventional DCs are recruited early to the skin upon in vivoallergen challenge. Immunouorescence staining and data analysiswere perormed as described in Figure . Counterstaining was perormed with Hoechst (blue, column ). CDCs were stained with amonoclonal antibody against BDCA- and envisioned with a secondary goat anti-mouse antibody conjugated with AF (red, column ).Column is the result o merging columns and . Representative microphotographs () o cDCs are shown h afer the rst challengeat the diluent (a) and allergen sites (b) and afer the sixth challenge at the allergen site (c). issue cDCs signicantly increased h afer therst allergen challenge compared to the diluent, and their numbers remained high afer the sixth allergen challenge, although the differencewas not signicant (d). A signicant inverse correlation was ound between blood and skin tissue-inltrating cDCs h afer the rst allergenchallenge ( = 0.0202; = 0.667) (e). Data are expressed as median with interquartile range (rst and third quartiles). < 0.05, WBC:whole blood cells.


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F : Activin-A is increased in the skin afer allergen challenge and correlates with BDCA+ cDCs. Activin-A in the skin was analysedusing the alkaline phosphatase-antialkaline phosphatase method (red colour). (a) Activin A in normal skin (diluent site) was minimal andmostly located in a scattered ashion in the basal cells o the epidermis (black arrows) with minimal expression in the dermis (red arrows)(). (b) Afer the rst allergen challenge, activin-A was more prominent and intense in the epidermis (black arrows) and in inltratinginammatory cells (red arrows) in the dermis (), also shown in (c) at higher magnication (). (d) Quantication o activin-A+

cells showed signicantly higher expression at the allergen site h afer the rst challenge compared to the diluent site. (e) Te numbers oactivin-A+ cells correlated with the numbers o BDCA-+ cDCs in the dermis h afer allergen challenge ( = 0.0219; = 0.662). Data are

expressed as median with interquartile range (rst and third quartiles).

< 0.001.

not signicant according to post hoc analysis (Figure (d)).Importantly, we observed that the percentages o circulatingcDCs inversely correlated with those o skin-inltrating cDCs( = 0.0202; = 0.6667,Figure (e)). No correlations wereobserved between DC subset numbers at the skin site and themagnitude o the early- or late-phase skin reactions (data notshown). In summary, these data provide evidence that cDCsare summoned to the inamed skin site early ollowing in vivoallergen exposure.

.. Activin-A Is Increased in the Skin afer Repeated InVivo Allergen Challenges and Correlates with cDC Numbers.Our data showed that activin-A expression in normal skin(diluent site) was minimal and mostly present in a scatteredashion at the basal cells o the epidermis with very lowlevelsobserved in the dermis (Figure (a)). Interestingly, aferthe rstin vivoallergen skin challenge, activin-A expressionwas more prominent and intense in the epidermis, as wellas, in inltrating inammatory cells in the dermis (Figures(b)and(c)). Activin-A was also observed at low levels inthe connective tissue. Following the sixth allergen challenge,activin-A in the epidermis and dermis at the allergen site

was higher compared to the diluent site, but to a lesserextent, and intensity compared to that afer the rst allergenchallenge (Figure (d)). As cDCs showed a similar kineticpattern to that o activin-A ollowing in vivo allergen chal-lenge, we investigated the existence o possible correlations.Interestingly, the number o activin-A+ cells correlated withthe number o BDCA-+ cDCs at the h time-point aferallergen challenge ( = 0.0219; = 0.662, Figure (e)).ogether, these ndings suggest a role or activin-A at theinamed skin site duringin vivoallergen challenge.

.. Activin-A Modulates the Chemokine Receptor Proleo Allergen-Stimulated CDc+ DCs. Previous studies havedemonstrated that human cDCs express higher levels oactivin-A type I andII signallingreceptors compared to pDCsandrespond actively to theligand [].Inviewoourndingsshowing a strong correlation between activin-A expressionand cDC trafficking at the inamed skin site upon allergenencounter, we hypothesized that activin-A may affect thecDC chemotactic prole. o address this, we examined theeffects o activin-A on chemokine receptor expression bycDCs isolated rom the peripheral blood o atopics during


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CCR4 CCR10 CCR

FSFS FS FS FS

6 CCR9 CXCR3

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14.2 3.39 10.5 4.84 3.23

10.8 7.37 11.5 4.45 3.27

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Der p1

Der p1 + activin-A

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0.0

(b)

F : Activin-A modies the chemokine receptor prole o CDc+ cDCs towards a skin-homing phenotype. CDc+ cDCs were isolatedrom the peripheral blood o individuals with atopy toDermatophagoides pteronyssinus (Der p), cultured or h with g/mL Der p in thepresence o ng/mL recombinant activin-A or PBS (control). DCs were stained with uorochrome-conjugated antibodies against humanCCR, CCR, CCR, CCR, and CXCR and analysed by ow-cytometry. (a) Activin-A induced an increase in CCR levels, concomitantwith a decrease in CCR on CDc+ cDCs during stimulation with Der pin vitro. No differences were observed regarding the expression oCCR, CCR, and CXCR in Der p-stimulated CDc+ cDCs in the presence or absence o activin-A. (b) Activin-A signicantly increased

the ratio o CCR/CCR expressing cDCs. Data are representative o two independent experiments. < 0.05.

stimulation with allergenin vitro. No signicant differenceswere observed in the levels o CXCR, CCR, or CCRon cDCs upon activin-A treatment (Figure (a)). Activin-A,however, increased the expression o CCR on CDc+ DCs,concomitant with a decrease in CCR, as compared to control(PBS) (Figure (a)). In act, activin-A signicantly increasedthe ratio o CCR/CCR expressing cDCs (Figure (b)).Chemokine receptor levels were very low in nonstimu-lated cDCs (medium alone) and increased upon LPS treat-ment (data not shown). Overall, these data suggest that

activin-A may be involved in retaining cDCs in the skinafer allergen challenge through the regulation o CCR andCCR expression.

4. Discussion

Human DC kinetics have been examined in the blood[, ],induced sputum [], or bronchial tissue [] o asthmatics,ollowing a single allergen inhalation or segmental allergen


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challenge. Still, simultaneous evaluation o DC kinetics atthe periphery and the challenged tissue site has not beeninvestigated. A single study has examined DC kinetics inthe blood and bronchoalveolar lavage uid (BALF) in sevenasthmatics afer a single segmental allergen challenge but didnot evaluate DC numbers at the inamed bronchial tissue

[]. Jahnsen et al. investigated DC kinetics in the peripheryand the challenged nasal tissue, but the study was limited topDCs []. o our knowledge, this is the rst human studyevaluating both cDC and pDC kinetics concomitantly in theperiphery and at the inamed tissue site afer repeated in vivoallergen skin challengesin atopic subjects, encapsulating boththe initial and chronic stages o allergic inammation.

Experimental skin allergen challenge has been widelyused to examine the cellular processes associated with early-and late-phase allergic reactions [] and to study the effectso repeated allergen exposure in the airways []. Moreover,these models imitate the chronicity o allergen exposure in amore standardized ashion and, as late-phase skin reactionsexhibit similar histopathological inammatory patterns tothat o the airways [], they can be used to recapitulatechronic allergic airway inammation []. Furthermore,these models demand only atopic status as a prerequisite,which usually requires no treatment. In asthma, however,treatment with corticosteroids is ofen warranted, which canact as a conounding actor by decreasing pDC absolute num-bers and cytokine responses uponin vivobronchial allergenchallenge []. Moreover, steroid treatment has been recentlyshown to increase the capacity o GILZ+ circulating DCsto activate allergen-specic IL- regs in allergic patients[]. In addition, the difficulties and increased patientrisk associated with perorming multiple bronchial allergenchallenges in conjunction with consecutive endobronchialbiopsies represent critical restraining actors. Still, our studypresents certain limitations, mainly pertaining to the act thatwe did not perorm challenges with an irrelevant allergen nordid we include healthy nonatopic controls to conrm that ourndings were due to allergen exposureper se.

Tere has been contradicting evidence pertinent to theeffects o repeated allergen challenges in the nose, airways,and skin o atopics. In certain studies, multiple challenges ledto tolerancewhile in othersled to enhanced inammatory cellmigration []. Te time interval between skin challenges,the type o allergen used, and the dosing scheme is crucial indetermining priming versus tolerance. Intervals longer thanthan one week seem to induce small late-phase reactions

[], while challenges with house dust mite ofen result in anincrease in late skin reactions compared to grass pollen [].We observed early- and late-phase skin reactions afer everychallenge with house dust mite and detected no differencesbetween challenges, as also shown by other investigators [].Tese data suggest that the possibility o desensitization isminimal.

Plasmacytoid DCs are primarily observed in peripheralblood, but they also migrate to lymphoid organs or tissuesduring inammation []. Te nding that there were nodifferences in the numbers o skin pDCs afer repeatedallergen challenges was rather unexpected. Previous stud-ies o bronchial allergen challenge in asthmatics have also

ailed to show an accumulation o pDCs in the bronchialtissue []. One could argue that single allergen challengemodels could be insufficient or the recruitment o pDCs;still, we did not observe signicant changes in pDCs evenafer six weekly allergen challenges, although a trend wasdetected. Te observation that pDC levels were decreased

in the periphery within one hour afer allergen challenge issuggestive o a rapid recruitment o these cells to the exposedskin site, possibly occurring long beore h. Moreover,pDCs have been shown to respond selectively to homeostaticchemokines, demonstrating an inherent tendency to migrateto secondary lymphoid organs rather than to inammatorytissue sites []. It is, thereore, possible that peripheral pDCswere recruited predominantly to local draining lymph nodes(DLNs).

Conventional DCs, in contrast to pDCs, circulate in theblood or reside in peripheral tissues [] and are quicklydepleted rom the circulation ollowing allergen inhalationin asthmatics []. Upon local allergen challenge, cDCshave been reported to accumulate within the bronchialmucosa []. It is, however, not clear whether this is dueto an enhanced cDC migration rom the periphery, thedifferentiation o DCs in situ, or decreased migration otissue-resident DCs towards DLNs upon allergen encounter[]. Our ndings demonstrate that cDCs accumulate atthe inamed skin site within h afer local allergen chal-lenge and inversely correlate with their numbers in theperiphery at the same time-point. Tese data support thenotion that cDCs are recruited rom the bloodstream tothe skin early ollowing allergen exposure. Still, this doesnot preclude the possibility o local DC differentiation romprecursor populations. In act, increased numbers o CD+

progenitor cells have been noted in the airways o asthmatics[]. In accordance with our ndings, cDCs have alsobeen reported to decrease in the periphery and increasein BALF h afer segmental allergen challenge in mildasthmatics; however, possible correlations werenot examined[]. Corrigan et al. also reported increased numbers oCc+ BDCA-+ cDCs bearing the thymic stromal lym-phopoietin receptor in the skin, h afer allergen challenge,and their data supported that these cells were recruitedrom the circulation rather being differentiated de novo[].

In order to investigate tissue mDCs, we used BDCA-as a cellular marker in the skin. CDa has also been usedto identiy cDCs in normal skin; however, CDc (BDCA-

) is considered a more useul marker as it colocalizeswith nearly all CDc+ cells [,]. Upon inammation, anadditional population o inltrating inammatory DCs hasbeen reported that is different rom steady state dermalcDCs and is considered their potential precursor [, ].In the acute phase o atopic dermatitis, these cells coex-press CDb/c and produce chemokines that attract H2cells[, , ]. It is unknown whether the cDCs examinedin the present studies contain also a population o thesehighly inammatory DCs. Delineation o the precise phe-notypic and unctional properties o cDCs recruited to theskin upon in vivo allergen challenge will help resolve thisquestion.


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Our results also demonstrate that activin-A is greatlyincreased in the skin aferin vivoallergen challenge. Impor-tantly, its expression correlates with the numbers o inltrat-ing cDCs at h afer challenge, suggesting that activin-Amay be involved in cDC recruitment to the skin. Activin-Ainduces DC migration and can act as a differentiating actor

or blood cDCs and lung Langerhans cell-like DCs [,].Although cDCs express both type I and II receptors [],activin-A has not been shown to exert direct chemotacticeffects on these cells. Still, activin-A alters the chemokineand the chemokine receptor prole o human Langerhansdendritic cells [, ]. In agreement, we demonstrate thatactivin-A induced a reciprocal shif up on CCR, with ashif down on CCR on allergen-stimulated CDc+ cDCsobtained rom atopic subjects. Both receptors are involved inDC and cell trafficking to the skin and inamed epithelia,including the lungs []. An inversion o the old increaseratio in CCR/CCR expression in skin allografs has beenpreviously associated with cell trafficking and allografrejection []. Moreover, a recent single study has shownthat circulating pDCs express increased levels o CCRand CCR in patients with allergic asthma []. ogether,these ndings prompt the speculation that activin-A may beinvolved in retaining cDCs at the inamed skin site, at leastpartly, through modulating CCR/CCR expression.

In summary, our study highlights key differences in thetrafficking o human pDCs and cDCs uponin vivoallergenchallenge in the skin, pointing to distinct roles in the allergicresponse. Importantly, our ndings acilitate the understand-ing o human DC behaviour during allergen encounter invivo, which is essential or controlling the balance betweenimmunity and tolerance in allergic diseases.

Abbreviations

AD: Atopic dermatitisBALF: Bronchoalveolar lavage uidBDCA: Blood dendritic cell antigenCCR: C-C moti chemokine receptorcDC: Conventional dendritic cellCXCR: CXC moti chemokine receptorDC: Dendritic cellDer p: Dermatophagoides pteronyssinusDLN: Draining lymph nodeEPR: Early-phase reaction

HDM: House dust miteIF: ImmunouorescenceIHC: ImmunohistochemistryLC: Langerhans cellLPR: Late-phase reactionLPS: LipopolysaccharidepDC: Plasmacytoid dendritic cellSP: Skin prick test.

Conflict of Interests

Te authors declare that there is no conict o interestsregarding the publication o this paper.

Authors Contribution

Stelios Vittorakis and Konstantinos Samitas recruited sub-jects, conducted skin challenges, perormed immunouo-rescence/conocal imaging experiments, evaluated biopsyspecimens, and wrote the paper. Soa ousa, Maria Agge-lakopoulou, and Maria Semitekolou perormed the in vitroexperiments and FACS analyses. Elefherios Zervas, and VilyPanoutsakopoulou contributed to the study design, assessedand interpreted experimental results, and reviewed the paper.Konstantinos Samitas and Elefherios Zervas reviewed allnal data and perormed statistical analyses. Georgina Xan-thou andMina Gaga denedthe research theme, designed theexperiments, contributed to the assessments and interpreta-tion o results, and wrote the paper. All authors have read andapproved the nal paper. Stelios Vittorakis and KonstantinosSamitas contributed equally.

Acknowledgments

Te authors are grateul to all subjects participating in thepresent study. Te authors would also like to thank SpyrosSpiridakis (Beckman Coulter, Greece) or his technical adviceon FACS experiments and analyses and Anna Agapaki, EleniRigana, and Stamatis Pagakis (Histochemistry Unit and Bio-logical Imaging Facility, Biomedical Research Foundation othe Academy o Athens) or their expert technical assistancewith conocal imaging.

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