Skin Barrier Dysfunction and Low Antimicrobial Peptide ...of AMPs in atopic dermatitis skin do not reach sufﬁcient levels to provide adequate control of cutaneous microbial colonization

Biology of Human Tumors

Skin Barrier Dysfunction and Low Antimicrobial PeptideExpression in Cutaneous T-cell Lymphoma

Hiraku Suga, Makoto Sugaya, Tomomitsu Miyagaki, Hanako Ohmatsu, Makiko Kawaguchi,Naomi Takahashi, Hideki Fujita, Yoshihide Asano, Yayoi Tada, Takafumi Kadono, and Shinichi Sato

AbstractPurpose:Atopic dermatitis is characterizedbydecreased expressionof filaggrin and loricrin. Patientswith

atopic dermatitis often suffer from skin infections, which are also frequently seen in patients with cutaneous

T-cell lymphoma (CTCL). In this study, we aimed to investigate the skin barrier in CTCL.

Experimental Design:We assessed skinmoisture and transepidermal water loss (TEWL) in patients with

CTCL.Wenext examinedmRNAexpression levels of filaggrin, loricrin, and antimicrobial peptides (AMP) in

skin samples of CTCL, using skin from healthy volunteers and patients with atopic dermatitis or psoriasis as

controls. Immunostainings for filaggrin, loricrin, and S100 proteins were also performed.

Results: Lower levels of skinmoisture accompanied byhigher levels of TEWLwere seen in lesional skin of

CTCL than in normal skin. CTCL lesional skin contained lower levels of filaggrin and loricrin mRNA than

normal skin, which was also true with atopic dermatitis and psoriatic skin. mRNA expression levels of

filaggrin in CTCL skin negatively correlated with disease severity markers. Expression levels of AMPs in

lesional skin of CTCL and atopic dermatitis were significantly lower than in psoriatic skin. Immunohis-

tochemistry confirmed decreased expression of filaggrin and loricrin in CTCL, atopic dermatitis, and

psoriatic skin and enhanced expression of S100 proteins in psoriatic skin.

Conclusions:Our results show that there is barrier dysfunction in CTCL skin, similar towhat is seenwith

atopic dermatitis skin. In addition, lowAMPexpression inCTCL skinwasdocumentedwhen comparedwith

psoriatic skin, which may explain frequent infections that can occur in patients with CTCL. Clin Cancer Res;

20(16); 4339–48. �2014 AACR.

IntroductionMycosis fungoides and S�ezary syndrome are the most

common types of cutaneous T-cell lymphoma (CTCL).Patients with mycosis fungoides typically have a pro-longed clinical course and only limited cases progressover years through patch, plaque, and tumor stages,followed by lymph node and visceral involvement (1).S�ezary syndrome is characterized by the triad of general-ized erythroderma (defined as affecting >80% of totalbody surface area), lymphadenopathy and presence ofmore than 1,000 per mm3 circulating atypical T cells withcerebriform nuclei, so-called S�ezary cells (2). Individualswith S�ezary syndrome usually show rapid disease pro-gression compared with patients with mycosis fungoides.Most cases with mycosis fungoides/S�ezary syndrome,

especially at an advanced stage, show a T helper 2(TH2)-dominant phenotype, characterized by increasedIL4, IL5, IL10, and IL13 production (3, 4). Patients withCTCL often suffer from skin infections with bacteria,viruses, and fungi as well. Among them, Staphylococcusaureus is the most common infection in CTCL (5). Inimmunocompromised patients with advanced-stageCTCL, skin infections occasionally lead to sepsis, multipleorgan failure, and death (6, 7). The underlying basis forthese infections has not been elucidated.

The epidermis provides an important physical barrieragainst environmental insults. Dry skin can cause manyserious complications such as discomfort and itching,development of dermatitis, and bacterial and viral infec-tions. Epidermal proteins such as filaggrin and loricrin areimportant in maintaining skin barrier function. Filaggrinaggregates keratin filaments and provides a cytoskeleton forthe cornified envelope (8). Loricrin is initially expressed inthe granular layer and comprises 70% of the total proteinmass of the cornified layer (8, 9). Skin barrier dysfunction isquite commonly seen in patients with atopic dermatitis,which is caused by loss of function in these proteins(10, 11).

Antimicrobial peptides (AMP) are part of the innateimmune response and are found among all classes of life

Authors' Affiliation: Department of Dermatology, Faculty of Medicine,University of Tokyo, Tokyo, Japan

Corresponding Author: Makoto Sugaya, Department of Dermatology,Faculty of Medicine, University of Tokyo, 7-3-1 Hongo, Bunkyo-ku, Tokyo113-8655, Japan. Phone: 81-3-5800-8661; Fax: 81-3-3814-1503; E-mail:[email protected]

doi: 10.1158/1078-0432.CCR-14-0077

�2014 American Association for Cancer Research.

ClinicalCancer

Research

www.aacrjournals.org 4339

on March 4, 2021. © 2014 American Association for Cancer Research. clincancerres.aacrjournals.org Downloaded from

Published OnlineFirst June 11, 2014; DOI: 10.1158/1078-0432.CCR-14-0077

http://clincancerres.aacrjournals.org/

(12). These peptides kill bacteria, mycobacteria, envel-oped viruses, and fungi. S100 family and human bdefensin (hBD)-1, 2, and 3, which are all representativeAMPs expressed by keratinocytes, are reported to beincreased in psoriatic skin, probably due to increasedexpression of IL17A and IL22 (13–15). It is still contro-versial whether AMPs are increased or decreased in atopicdermatitis lesional skin (16–19). Although there havebeen considerable numbers of articles showing increasedAMP expression in atopic dermatitis lesional skin, recentreports indicate that induction, release, and mobilizationof AMPs in atopic dermatitis skin do not reach sufficientlevels to provide adequate control of cutaneous microbialcolonization (20). TH2 cytokines appear to negativelyinfluence the expression and induction of some AMPs(18, 19). Interestingly, a recent report shows that pro-duction of AMPs by keratinocytes in adult T-cell leuke-mia/lymphoma is reduced, leading to perturbed innateimmunity and the frequent occurrence of superficialdermatophytosis (21).

In this study, we investigated skin barrier function andexpression of AMPs in patients with CTCL, which sharesmany common immunologic features with atopic derma-titis. Specifically, we assessed skinmoisture, transepidermalwater loss (TEWL), and expression of filaggrin, loricrin,S100 proteins, and defensins in patients with CTCL. Skinfrom healthy volunteers and patients with atopic dermatitisor psoriasis were used as control samples. Taken together,our results suggest that skin barrier dysfunction occurs inpatients with CTCL, similar to what is seen in patients withatopic dermatitis. These cutaneous defects likely explain thefrequent occurrence of infections in patients with CTCL andmay eventually lead to new strategies to improve disease-associated morbidity.

Materials and MethodsPatients and samples

mRNA was obtained from biopsy materials of lesionalskin of CTCL (n ¼ 26, 17 males and 9 female; mean � SD:age: 57.6�12.3 years; 7 caseswithpatchmycosis fungoides,8 cases with plaque mycosis fungoides, 5 cases with tumormycosis fungoides, 2 cases with erythrodermic mycosisfungoides, and 4 cases with S�ezary syndrome), atopic der-matitis (n¼ 6, all extrinsic type), and psoriasis (n¼ 5), andnormal skin adjacent to benign skin tumors (n ¼ 6) usingRNeasy Fibrous TissueMiniKit (QIAGEN).All patientswereeither untreated or treated with only topical corticosteroidsat the time of biopsy. The diagnosis of mycosis fungoidesand S�ezary syndrome and the stages of CTCL were based onclinical criteria as well as on histologic and IHC assessmentaccording to World Health Organization classification andthe criteria of the International Society for Cutaneous Lym-phomas (2, 22).When classifying patients into patchmyco-sis fungoides, plaque mycosis fungoides, tumor mycosisfungoides, or erythrodermic mycosis fungoides/S�ezary syn-drome, the most severe skin lesion was taken into consid-eration. Atopic dermatitis was diagnosed according toHani-fin and Rajka criteria (23). Healthy controls had no historyof CTCL, atopic dermatitis, psoriasis, or any inflammatoryskin diseases. Indeed, we specifically excluded patientsunder treatment for various internal and inflammatorydisorders other than CTCL, atopic dermatitis, and psoriasis.All samples were collected after informed consent duringdaily clinical practice. The medical ethical committee of theUniversity of Tokyo (Tokyo, Japan) approved all describedstudies, and the study was conducted according to theDeclaration of Helsinki Principles.

Skin moisture and TEWLWe evaluated skin moisture and TEWL in lesional skin of

patients with CTCL (n¼ 11, 8 males and 3 females; 59.4 �11.3 years; 4 cases with patch mycosis fungoides, 2 caseswith plaquemycosis fungoides, 3 cases with tumormycosisfungoides, and 2 cases with erythrodermic mycosis fun-goides/S�ezary syndrome) and healthy volunteers (n ¼ 8, 6males and 2 females; 54.4� 20.3 years). Skin moisture andTEWL were evaluated at least 24 hours after the last appli-cation of moisturizers and/or topical corticosteroids aspreviously described (24, 25). CTCL lesions located in thelower abdominal area or equivalent normal skin areas inhealthy volunteers were chosen as the test regions. More-over, we examined skin moisture and TEWL in perilesionalnormal-appearing skin in the same patients with CTCL.Perilesional skin was located at least 3 cm outside of theborder ofCTCL lesional skin.Noneof patientswithCTCL inthis study had ichthyosis vulgaris. The measurements wereperformed in the months of April and May, when thehumidity was 40% to 50% and the room temperature waskept at 20�C. First, we put water-soaked gauze on the testregion. Next, we softly wiped the regionwith dry gauze. Fiveminutes later, we evaluated skin moisture and TEWL byusing SKICON-200EX (IBS Co. Ltd.) and Tewameter TM300 (Courage&Khazaka), respectively. Skinmoisture levels

Translational RelevanceSkin is a barrier against pathogens such as bacteria,

viruses, and fungi. Atopic dermatitis is characterized bydecreased skin expression of filaggrin and loricrin, lead-ing to abnormalities in skin barrier function andincreased susceptibility to infections. Patients with cuta-neous T-cell lymphoma (CTCL), which is a T helper 2–dominant disease like atopic dermatitis, often sufferfrom skin infection as well, leading us to investigate theskin barrier in CTCL. In this study, we have shown thatmRNA levels of filaggrin and loricrin in lesional skin ofadvanced CTCL are decreased compared with normalskin, which negatively correlate with disease severitymarkers such as CCL17 and IL22. Antimicrobial peptideexpression in lesional skin of CTCL was significantlylower than in psoriatic skin. Identification of skin barrierdysfunction in patients with CTCL, as shown here, maypotentially lead to therapeutic strategies to prevent infec-tions in these patients, leading to improved disease-associated morbidity.

Suga et al.

Clin Cancer Res; 20(16) August 15, 2014 Clinical Cancer Research4340




were displayed on the machine within 1 second afterapplication of the probe onto the skin. Regarding TEWLlevels, values were displayed on a recorder, and the meanvalue during the period 40 seconds after application of theprobe onto the skin was calculated. Skin moisture levelswere evaluated 5 times in one skin lesion, and TEWL valueswere measured twice. Mean values were calculated.

Quantitative reverse transcriptase PCR assaycDNA was synthesized using iScript cDNA Synthesis Kit

(Bio-Rad Laboratories). Quantitative reverse transcriptasePCR was performed as described previously based on SYBRGreen assay (26). Primers for human filaggrin, loricrin,S100A7, S100A8, S100A9, hBD-1, hBD-2, hBD-3, andGAPDH were as follows: filaggrin: forward, 50-GAA GACAAG GAT CGC ACC AC-30 and reverse, 50-ATG GTG TCCTGA CCC TCT TG-30; loricrin: forward, 50-TCA TGA TGCTACCCGAGGTTTG-30 and reverse, 50-CAGAAC TAGATGCAG CCG GAG A-30; S100A7: forward, 50-CTT CCT TAGTGC CTG TGA CAA AAA-30 and reverse, 50-AAA GAC AGAAACTCAGAAAAA TCAATC T-30; S100A8: forward, 50-ATGCCG TCT ACA GGG ATG AC-30 and reverse, 50-ACG CCCATC TTT ATC ACC AG-30; S100A9: forward, 50-CAG CTGGAA CGC AAC ATA GA-30 and reverse, 50-TCA GCT GCTTGT CTG CAT TT-30; hBD-1: forward, 50-AGA TGG CCTCAG GTG GTA ACT TT-30 and reverse, 50-GGG CAG GCAGAA TAG AGA CAT T-30; hBD-2: forward, 50-GAT GCC TCTTCC AGG TGT TTT T-30 and reverse, 50-GGA TGA CAT ATGGCT CCA CTC TT-30; hBD-3: forward, 50-GTG AAG CCTAGC AGC TAT GAG GAT-30 and reverse, 50-TGA TTC CTCCATGACCTGGAA-30; GAPDH: forward, 50-ACCCAC TCCTCC ACC TTT GA-30 and reverse, 50-CAT ACC AGG AAATGA GCT TGA CAA-30.

ImmunohistochemistryWe performed IHC staining for filaggrin, loricrin,

S100A7, and S100A8 with lesional skin of 20 cases of CTCL

[patch mycosis fungoides (n ¼ 5), plaque mycosis fun-goides (n ¼ 5), tumor mycosis fungoides (n ¼ 5), erythro-dermicmycosis fungoides/S�ezary syndrome (n¼5)], atopicdermatitis (n ¼ 5), and psoriasis (n ¼ 5). Normal skinadjacent to benign skin tumors served as controls (n ¼ 5).Briefly, 5-mm-thick tissue sections from formaldehyde-fixedand paraffin-embedded samples were dewaxed and rehy-drated. These sections were then stained with mouse anti-human filaggrin monoclonal antibody (Santa Cruz Bio-tech), rabbit anti-human loricrin polyclonal antibody(Santa Cruz),mouse anti-human S100A7monoclonal anti-body (Santa Cruz), or mouse anti-human S100A8 mono-clonal antibody (Santa Cruz), followed by ABC staining(Vector Lab). Diaminobenzidine was used for visualizingthe staining, and counterstaining with Mayer hematoxylinwas performed, according to manufacturers’ instructions.

Statistical analysesStatistical analyses were performed using the Mann–

Whitney U test and the Student t test for comparison of 2groups. For testing equality of population means among 3or more groups, the Kruskal–Wallis test and the Scheffe Ftest were used. Correlation coefficients were determined byusing the Spearman rank correlation test. P < 0.05 wasconsidered statistically significant.

ResultsLower levels of skin moisture accompanied by higherlevels of TEWL in lesional skin of CTCL

To investigate skinbarrier function inpatientswithCTCL,we evaluated skin moisture and TEWL in lesional andnonlesional skin ofCTCL and innormal skin. Skinmoisturelevels in CTCL lesional skin were significantly lower thanthose found in nonlesional skin and normal skin (Fig. 1, P <0.01, each). Moreover, skin moisture levels in nonlesionalskin from patients with CTCL were lower than in normalcontrol skin (Fig. 1, P < 0.01).

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Figure 1. Skin moisture and TEWLin lesional and nonlesional skinfrom patients with CTCL and innormal control skin. The measuredvalues from individual patientswere plotted by dots. �, P < 0.05;��, P < 0.01.

Skin Barrier Dysfunction in CTCL

www.aacrjournals.org Clin Cancer Res; 20(16) August 15, 2014 4341




TEWL values in CTCL lesional skin were significantlyhigher those in nonlesional skin and normal skin (Fig. 1,P < 0.01, each). There were also significant differences inTEWL values between nonlesional CTCL skin and normalcontrol skin (Fig. 1, P < 0.05). Thus, skin barrier functionwas abnormal in both lesional and nonlesional skin ofpatients with CTCL when compared with normal controlskin.

Decreased mRNA expression of filaggrin and loricrincombined with increased mRNA expression of S100family in CTCL skin

We next analyzed expression levels of skin barrier–asso-ciated proteins and AMPs in lesional skin of CTCL, atopicdermatitis, psoriasis, and healthy individuals. FilaggrinmRNA expression levels in lesional skin of plaque, tumor,or erythroderma of CTCL were significantly decreased com-pared with normal skin (Fig. 2). Similar results wereobtained with regard to loricrin mRNA. Filaggrin and lor-icrin mRNA expression levels in lesional skin of atopicdermatitis and psoriasis were also decreased comparedwith

normal controls (Fig. 2), which was consistent with previ-ous reports (10, 11). S100A7 mRNA expression levels inlesional skin of patch, plaque, tumor, erythroderma ofCTCL, atopic dermatitis, and psoriasis were significantlyhigher than in normal controls (Fig. 2; P < 0.05, P < 0.01,P < 0.05, P < 0.01, P < 0.05, and P < 0.01, respectively).Similarly, lesional skin of plaque, tumor, erythroderma ofCTCL, atopic dermatitis, and psoriasis expressed significanthigher levels of S100A8 mRNA than in normal skin (Fig. 2;P < 0.01, P < 0.05, P < 0.05, P < 0.05, and P < 0.01,respectively). S100A9 mRNA expression levels in lesionalskin of plaque, tumor of CTCL, and psoriasis were signif-icantly higher than in normal controls (Fig. 2; P < 0.01, P <0.05, and P < 0.05, respectively). With regard to hBD-1, inlesional skin of plaque, tumor, erythroderma of CTCL,and atopic dermatitis, significantly decreasedmRNAexpres-sion levels were detected compared with normal controls(Fig. 2; P < 0.05, each). Although previous reports showedthat hBD-1 expression correlated with disease activity inpsoriasis (15, 27), it was not elevated in lesional skin ofpsoriasis comparedwith normal skin.Of note,mRNA levels

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Figure 2. mRNA expression of filaggrin, loricrin, S100A7, S100A8, S100A9, and hBD-1 in lesional skin of patch, plaque, tumor, erythroderma of CTCL, atopicdermatitis (AD), and psoriasis and in normal skin. Each histogram shows themeanþ SEM. �, P < 0.05; ��, P < 0.01 versus normal skin; †,P < 0.05; ††,P < 0.01versus psoriasis.

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of S100A7 and S100A8 in CTCL lesional skin were signif-icantly decreased as compared with levels in psoriatic skin(Fig. 2; P < 0.01, each). There were no significant differencesinhBD-2 andhBD-3 expression among the groups (datanotshown). Thus, filaggrin and loricrinmRNAexpression levelswere decreased in CTCL lesional skin combined withincreased S100 family protein expression, although thelatter was not as high as seen in psoriatic skin.

Correlations between filaggrin expression in lesionalskin of CTCL and disease activityWe evaluated correlations between expression levels of

filaggrin and those of loricrin, S100 family, hBD-1, hBD-2,hBD-3, and disease severity markers in CTCL lesional skin.We have previously reported that expression levels ofCCL17, CCL18, CCR4, IL4, and IL22 are associated withdisease progression in CTCL (3, 28–31). LIGHT[lymphotoxin-like, exhibits inducible expression, and com-petes with HSV glycoprotein D for herpesvirus entry medi-ator (HVEM), a receptor expressed by T lymphocytes] is alsocorrelated with disease progression in CTCL, whereasHVEM expression in CTCL skin negatively correlates withdisease progression (28). Filaggrin expression levels posi-tively correlated with those of loricrin (Fig. 3A), which isconsistent with the fact that both molecules are barrierfunction–related proteins downregulated by TH2 cytokines(32, 33). Filaggrin expression levels negatively correlatedwith those of CCL17, CCL18, CCR4, IL4, IL22, and LIGHTand positively correlated with those of HVEM (Fig. 3A),suggesting a significant negative correlationbetweendiseaseprogression inCTCL and filaggrin expression.Moreover, weevaluated associations between filaggrin expression inCTCL lesional skin and serum levels of sIL2R or CCL17(29, 31). Interestingly, there were significant negative cor-relations between filaggrin expression and serum levels ofthese disease markers (Fig. 3B). Thus, as severity of CTCLprogresses, expression of skin barrier–related proteinsdecreases in lesional skin.

Correlations between S100A7 expression in lesionalskin of CTCL and disease activityWe next evaluated correlations between expression levels

of S100A7, a representative AMP (34), and those of loricrin,S100A8, S100A9, hBD-1, hBD-2, hBD-3, and disease sever-ity markers in CTCL lesional skin. As expected, S100A8 andS100A9 expression levels positively correlated with S100A7expression levels (Fig. 4A). Expression levels of CCL26 andCCR3, which is a prototypic TH2 chemokine/chemokinereceptor pair, negatively correlated with those of S100A7(Fig. 4A). We have reported that expression levels of CCL26and CCR3 are increased in lesional skin of advanced CTCLcompared with normal controls (29, 35, 36). Thus, whendisease progresses, production of AMPs in CTCL lesionalskin, which is abundant in TH2 cytokines and chemokines,decreases. In turn, this likely leads to the frequent occur-rence of skin infections seen in patients with advancedCTCL.

No correlation between S100A7 and IL17A or IL22expression in CTCL lesional skin

Expression of AMPs, including S100A7, hBD-1, andhBD-2, by keratinocytes is increased by stimulation with IL17Aand/or IL22 in vitro (13, 14). Expression of these cytokines isupregulated in psoriatic skin compared with normal skin(37, 38). We have reported that IL22, but not IL17A, isupregulated in CTCL lesional skin (27). Therefore, weexamined correlations between S100A7 expression andIL17A or IL22 expression in CTCL lesional skin. There wereno positive correlations between expression levels ofS100A7 and either IL17A or IL22 in CTCL lesional skin(Fig. 4B). Thus, AMP expression was not increased inproportion to IL17A or IL22 expression in CTCL skin,probably because TH2 cytokines, abundantly expressed inadvanced CTCL skin, blocked AMP expression by lesionalkeratinocytes as was reported in vitro (18, 19).

Decreased filaggrin and loricrin expression bykeratinocytes in lesional skin of CTCL, atopicdermatitis, and psoriasis and enhanced S100A7 andS100A8 expression in psoriatic skin

IHC stainings for filaggrin, loricrin, S100A7, and S100A8were performed using lesional skin of patch, plaque, tumor,erythroderma of CTCL, atopic dermatitis, psoriasis, andnormal skin. In patch and plaque CTCL cases, filaggrin andloricrin expression by keratinocytes was decreased com-pared with normal skin (Fig. 5 and Table 1). In almost allcases with tumor and erythroderma of CTCL, atopic der-matitis, and psoriasis, filaggrin and loricrin expression wasremarkably decreased (Fig. 5 and Table 1). S100A7 andS100A8 expression was remarkably enhanced only in pso-riatic skin. These IHC findings were largely consistent withmRNA levels detected by real-time PCR (Fig. 2). Thus,expression of skin barrier–related proteins by keratinocytesin lesional skin of CTCL, atopic dermatitis, and psoriasiswas decreased, whereas AMPs were highly produced bykeratinocytes of lesional skin of psoriasis, but not in CTCLor atopic dermatitis skin.

DiscussionOur study revealed that skin moisture levels were

decreased, and TEWLwas increased in lesional skin of CTCLcompared with normal skin. Lesional skin of advancedCTCL also expressed lower levels of filaggrin mRNA, whichnegatively correlated with mRNA levels of disease severitymarkers. Expression levels of AMPs in lesional skin of CTCLand atopic dermatitis were significantly lower than those inpsoriatic skin,whichmay explain the frequent occurrence ofcutaneous infections in patients with CTCL.

Measuring skin moisture and TEWL are noninvasivemeans to assess barrier function of the stratum corneum.Previously, skin moisture and TEWL were evaluated withvarious skin diseases such as atopic dermatitis, ichthyosis,and psoriasis, all of which are strongly associated with dryskin (24, 25). To the best of our knowledge, this is the firststudy to evaluate skin moisture and TEWL in patients withCTCL.Ofnote, to control for any age-related changes in skin






function (39), we used normal volunteers of the approxi-mate same age as the patients with CTCL. Skin moisturelevels in lesional skin of CTCL were significantly lower thanthose in normal skin, whereas TEWL in CTCL skin wassignificantly higher than in normal skin (Fig. 1). Moreover,lower levels of skinmoisture combinedwith higher levels ofTEWL were detected in CTCL perilesional skin than innormal skin. Although we did not evaluate perilesionalskin histologically in this study, atypical T-cell infiltrationis often seen in normal-appearing skin of patients with

CTCL. Therefore, direct infiltration of tumor cells as well asTH2-dominant systemic inflammation can cause skin bar-rier dysfunction in nonlesional skin of CTCL. Thus, weclearly show here that patients with CTCL have dry skin,which can subsequently lead to pain, pruritus, andinfections.

The recent identification and confirmation of loss-of-function mutations in filaggrin as a major risk factor foratopic dermatitis sheds new light on the immunopathogen-esis of this disease (10, 11). Skin barrier dysfunction and

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Figure 3. Correlations between filaggrin expression in lesional skin of CTCL and disease activity. A, significant positive or negative correlations betweenexpression levels of filaggrin in CTCL lesional skin and those of loricrin or diseasemarkers. B, significant negative correlations between filaggrin expression inCTCL lesional skin and serum-soluble IL2 receptor or CCL17 levels. The measured values from individual patients were plotted by dots.

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resultant diminished epidermal defense mechanisms toallergens and microbes are regarded as early steps in theonset of atopic dermatitis (16–19). TH2-dominant immuneresponses such as increased levels of IgE may result from,rather than cause, refractory eczema as has been reported inmice repeatedly exposed to cutaneous allergens (40). A

recent report, however, showed that expression of filaggrinand loricrin by primary human keratinocytes was inhibitedby adding IL4, IL13, or the combination of IL4 and IL13(33). Skin biopsies from STAT6 transgenic mice were defi-cient in loricrin and involucrin, suggesting that TH2 cyto-kines such as IL4 and IL13 inhibit expression of barrier

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A

IL22

CC

R3

(rat

io to

GA

PD

H)

(rat

io to

GA

PD

H)

(rat

io to

GA

PD

H)

(rat

io to

GA

PD

H)

(rat

io to

GA

PD

H)

(rat

io to

GA

PD

H)


(ratio to GAPDH)(ratio to GAPDH)


S100A7 S100A7

S100A7 S100A7

S100A7 S100A7

(×10−1) (×10−1)

(×10−3) (×10−1)

r = 0.89

P < 0.01r = 0.83

P < 0.01

r = −0.69

P < 0.01r = −0.58

P < 0.01

r = −0.06

P > 0.05

r = −0.09

P > 0.05

A

B

Figure 4. Correlations betweenS100A7 expression in lesional skinof CTCL and disease activity. A,significant positive or negativecorrelations between expressionlevels of S100A7 and those ofS100A8, S100A9, CCL26, or CCR3in CTCL lesional skin. B, nosignificant correlations betweenexpression levels of S100A7 andthose of IL17A or IL22 in CTCLlesional skin. The measured valuesfrom individual patients wereplotted by dots.






function–related proteins through a STAT6-dependentmechanism (33). IL4 and IL13, which are highly expressedin skin andbloodof patientswith either atopic dermatitis orCTCL (30, 41, 42), may in turn downregulate filaggrin andloricrin expression in these patients. Indeed, we foundsignificant negative correlations between filaggrin expres-sion and disease severity markers (including IL4) in CTCLlesional skin (Fig. 3A). On the other hand, expression offilaggrin and loricrin was also decreased in lesional skin ofpsoriasis patients (Fig. 2), which is consistent with previousreports (13–15). TNFa, expressed in psoriatic skin, down-regulates filaggrin and loricrin expression in vitro. Ourresults, along with previous reports, suggest that decreasedexpression of filaggrin is not a specific finding of atopic

dermatitis, as this was readily seen in CTCL and psoriasispatients as well.

Whereas CTCL, atopic dermatitis, and psoriasis are all T-cell–associated diseases that share common features such asepidermal hyperplasia, abundant inflammatory cell infil-trates, and decreased expression of filaggrin, the 2 formerdiseases have different immune and barrier phenotypesfrom the latter. Atopic dermatitis is a TH2/TH22-polarizeddisease with an attenuated TH17 axis (43). Expressionof IL22 and TH2 cytokines such as IL4 and IL10, but notIL17A, is also elevated in lesional skin of CTCL (30). Incontrast, IL17A and IL22 are elevated in psoriatic skin,but not TH2 cytokines. TH2 cytokines inhibit secretion ofAMPs such as S100A7, hBD-2, and hBD-3 in primary

Normal Patch/plaque Tumor Erythroderma AD Psoriasis

Filaggrin

Loricrin

S100A7

S100A8

(++)

(++) (++)

(++)

(++)

(+)

(−) (−) (−)

(−)(−)

(−)(−)(−)(−)(−)

(−) (−) (−) (−) (−)

(+) (+)

(+)

CTCL

Figure 5. IHC staining for filaggrin, loricrin, S100A7, and S100A8 in lesional skin of patch, plaque, tumor, and erythroderma of CTCL, atopic dermatitis (AD),psoriasis and in normal skin (original magnification, �400). Representative pictures of 5 cases in each group. �, no expression; þ, modest expression; þþ,high expression.

Table 1. Filaggrin, loricrin, S100A7, and S100A8 expression in lesional skin of patch, plaque, tumor,erythroderma of CTCL, atopic dermatitis, psoriasis and in normal skin

filaggrin loricrin S100A7 S100A8

Diagnosis Cases � þ þþ � þ þþ � þ þþ � þ þþNormal 5 0% 40% 60% 0% 40% 60% 80% 20% 0% 80% 20% 0%CTCL (patch) 5 20% 60% 20% 40% 60% 0% 60% 40% 0% 40% 60% 0%CTCL (plaque) 5 60% 40% 0% 60% 40% 0% 60% 40% 0% 60% 20% 20%CTCL (tumor) 5 80% 20% 0% 80% 20% 0% 60% 40% 0% 40% 60% 0%CTCL (erythroderma) 5 80% 20% 0% 60% 40% 0% 80% 20% 0% 60% 40% 0%Atopic dermatitis 5 80% 20% 0% 80% 20% 0% 40% 60% 0% 60% 40% 0%Psoriasis 5 80% 20% 0% 80% 20% 0% 0% 40% 60% 0% 40% 60%

Suga et al.





keratinocytes (44–46). Consistently, in CTCL lesional skin,we detected significant negative correlations betweenexpression levels of S100A7 and those of CCL26 or CCR3,a representative TH2 chemokine/chemokine receptor pair(Fig. 4A). TH2 cytokines also suppress TH1 cytokines, whichare effective for antitumor immunity and infections (47,48). Thus, a TH2-dominant cytokine milieu downregulatesimmunity against infections, which are commonly seen inlesional skin of CTCL as well as in atopic dermatitis skin.Not surprisingly, patients with psoriasis do not have clinicalissues with skin infections, as TH2 cytokines are notdominant.We also showed that expression levels of AMPs were

higher in psoriatic skin than in CTCL or atopic dermatitisskin (Fig. 2). IL17A, together with IL22, induces AMPssuch as S100A7, S100A8, S100A9, and hBD-2 (49).Interestingly, there were positive correlations betweenexpression levels of S100A7 and those of IL17A or IL22in psoriatic skin (data not shown), whereas no suchcorrelations were found in CTCL skin (Fig. 4B). BecauseTH2 cytokines suppress expression of AMPs as well asIL17A, high expression of TH2 cytokines may be the mainreason why AMP levels were decreased in CTCL andatopic dermatitis compared with psoriasis. In the skin ofpatients with erythrodermic CTCL, Staphylococcus aureus–derived superantigen enterotoxins are commonly found,which could exacerbate chronic expansion of T cells,including T-cell receptor Vb2-bearing cells (5, 50). There-fore, insufficient induction of AMPs, especially S100A8and S100A9, may cause infection with staphylococcus inCTCL skin, which may induce expansion of T cells bear-ing specific T-cell receptors. Although psoriatic skin isoften colonized with Staphylococcus aureus, life-threaten-ing infection is rarely seen probably due to sufficientexpression of AMPs.

In conclusion, our study has revealed that skin barrierdysfunction is present in CTCL, similar to what has beenreported in atopic dermatitis. TH2/TH22-polarized immunestatus together with an attenuated TH17 axis may causedecreases in filaggrin expression and insufficient inductionof AMPs. Thus, improving skin barrier function by attenu-ating TH2 responses could lead to better control of cutane-ous infections, improving overall disease-associated mor-bidity in patients with CTCL.

Disclosure of Potential Conflicts of InterestNo potential conflicts of interest were disclosed.

Authors' ContributionsConception and design: M. Sugaya, H. FujitaDevelopment of methodology: H. Suga, T. MiyagakiAcquisitionofdata (provided animals, acquired andmanagedpatients,provided facilities, etc.):H. Suga, M. Sugaya, T. Miyagaki, H. Ohmatsu, N.Takahashi, H. FujitaAnalysis and interpretation of data (e.g., statistical analysis, biosta-tistics, computational analysis): H. Suga, M. Sugaya, M. Kawaguchi,H. Fujita, Y. AsanoWriting, review, and/or revision of themanuscript:M. Sugaya, H. Fujita,Y. Asano, Y. Tada, T. Kadono, S. SatoAdministrative, technical, or material support (i.e., reporting or orga-nizing data, constructing databases): Y. Asano, S. SatoStudy supervision: H. Fujita, Y. Asano, T. Kadono, S. Sato

AcknowledgmentsThe authors thankDr. AndrewBlauvelt (OregonMedical ResearchCenter,

Portland, OR) for many helpful comments and Tamami Kaga for technicalassistance.

Grant SupportThis work was supported by grants from the Ministry of Education,

Culture, Sports, Science and Technology (Japan).The costs of publication of this articlewere defrayed in part by the payment

of page charges. This article must therefore be hereby marked advertisementin accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

Received January 10, 2014; revised April 19, 2014; acceptedMay 16, 2014;published OnlineFirst June 11, 2014.

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Suga et al.




2014;20:4339-4348. Published OnlineFirst June 11, 2014.Clin Cancer Res Hiraku Suga, Makoto Sugaya, Tomomitsu Miyagaki, et al. Expression in Cutaneous T-cell LymphomaSkin Barrier Dysfunction and Low Antimicrobial Peptide

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Skin Barrier Dysfunction and Low Antimicrobial Peptide ...of AMPs in atopic dermatitis skin do not reach sufﬁcient levels to provide adequate control of cutaneous microbial colonization